NeuroToxicology 33 (2012) 391400

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NeuroToxicology

Review

Organophosphate-induced brain damage: Mechanisms, neuropsychiatric and neurological consequences, and potential therapeutic strategies
Yun Chen *
BrightstarTech, Inc., 23102 Meadow Mist Road, Clarksburg, MD 20871, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 January 2012 Accepted 25 March 2012 Available online 2 April 2012 Keywords: Organophosphates Nerve agents Acetylcholinesterase Cholinergic neuronal excitotoxicity Secondary neuronal damage Neuropsychiatric disorders Neurological impairments Neuroprotective strategies

Organophosphate (OP)-induced brain damage is dened as progressive damage to the brain, resulting from the cholinergic neuronal excitotoxicity and dysfunction induced by OP-induced irreversible AChE inhibition. This delayed secondary neuronal damage that occurs mainly in the cholinergic regions of the brain that contain dense accumulations of cholinergic neurons and the majority of cholinergic projection, might be largely responsible for persistent profound neuropsychiatric and neurological impairments (memory, cognitive, mental, emotional, motor and sensory decits) in the victims of OP poisoning. Neuroprotective strategies for attenuating OP-induced brain damage should target different development stages of OP-induced brain damage, and may include but not limited to: (1) Antidote therapies with atropine and related efcient anticholinergic drugs; (2) Anti-excitotoxic therapies targeting attenuation of cerebral edema and inammatory reaction, blockage of calcium inux, inhibition of apoptosis program, and the control of seizures; (3) Neuroprotective strategies using cytokines, antioxidants and NMDAR antagonists (a single drug or a combination of drugs) to slow down the process of secondary neuronal damage; and (4) Therapies targeting individual symptoms or clusters of chronic neuropsychiatric and neurological symptoms. These neuroprotective strategies may help limit or prevent secondary neuronal damage at the early stage of OP poisoning and attenuate the subsequent neuropsychiatric and neurological impairments, thus reducing the long-term disability caused by exposure to OPs. 2012 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cholinergic neuronal excitotoxicity and dysfunction resulting from irreversible inhibition of AChE by OPs . . . . . Secondary neuronal damage triggered by cholinergic neuronal excitotoxicity and dysfunction . . . . . . . . . . . . . . . Secondary neuronal damage and long-term neuropsychiatric and neurological disorders . . . . . . . . . . . . . . . . . . . Persistent memory and cognitive decits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. 4.2. Psychomotor performance decits and somatic complaints. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Non-specic mental and emotional symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Potential secondary damage effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. 4.4.1. Posttraumatic stress disorder (PTSD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Brain cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2. Potential therapeutic strategies for victims of OP poisoning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antidote therapies in the OP toxicity phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. 5.2. Anti-excitotoxic therapies in the cholinergic excitotoxicity phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neuroprotective strategies against secondary neuronal damage in the secondary neuronal damage phase 5.3. Therapies targeting individual symptoms or clusters in the development phase of chronic 5.4. neuropsychiatric and neurological symptoms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 392 392 394 394 395 395 395 395 396 396 396 397 397 398 398 398 398

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* Tel.: +1 301 318 3442; fax: +1 301 944 3548. E-mail address: chen.yun_bst@yahoo.com. 0161-813X/$ see front matter 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neuro.2012.03.011

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1. Introduction Organophosphate (OP) compounds are a group of highly toxic chemicals that have been used widely as pesticides and developed as chemical warfare nerve agents such as soman (GD), sarin (GB), tabun (GA), cyclosarin (GF) and VX. OPs can be efciently absorbed by inhalation, ingestion, and skin penetration. OP poisoning resulting from accidental or intentional (e.g. suicides by intentional ingestion of pesticides) exposure to pesticides is one of the most common poisonings worldwide, which causes approximately one million cases of poisoning each year with several hundred thousand deaths, mostly in developing countries (Pandit et al., 2011; Yurumez et al., 2007). Clinical manifestations may vary among victims of OP poisoning, depending on the specic compound, the amount, route, and length of time of exposure, as well as the age and health status of the person exposed. OPs disrupt the functioning of cholinergic nervous system by irreversibly inhibiting acetylcholinesterase (AChE), which is responsible for the hydrolysis (breakdown) of acetylcholine (ACh) in the synapse. The inhibition of AChE by OPs results in both the accumulation of ACh at synapses of the central and peripheral nervous systems and overstimulation of cholinergic receptors that exceeds normal physiological limits (Gallo and Lawryk, 1991). Acute, excessive stimulation of cholinergic receptors (primarily the muscarinic receptor, mAChR, in the brain) causes cholinergic neuronal excitotoxicity and dysfunction, which are largely responsible for the cholinergic crisis in the acute phase of the OP exposures (within minutes), and could subsequently cause secondary neuronal damage and chronic neuropsychiatric consequences (Wiener and Hoffman, 2004; Apland et al., 2010). During recent years the research on cholinergic effects of OPs in the brain has become more clinically oriented. Great attention has been paid especially to the involvement of cholinergic neuronal excitotoxicity and neurotransmission decits in the pathophysiology of secondary neuronal damage, and long-term neuropsychiatric and neurological consequences following exposure to OPs (Nordberg et al., 1990; Petras, 1994; Carpentier et al., 2008). 2. Cholinergic neuronal excitotoxicity and dysfunction resulting from irreversible inhibition of AChE by OPs AChE is an enzyme that terminates synaptic transmission by hydrolytic degradation of the ACh into the inactive products choline and acetic acid, at neuromuscular junctions and central cholinergic nervous system. ACh is the rst compound to be identied as a neurotransmitter in the central nervous system (CNS). Not all parts of CNS contain ACh; those areas that have high concentrations are the cortex, hypothalamus, amygdale, thalamus and anterior spinal roots. Anatomically, two major cholinergic regions have now been identied in the brain. The rst, and probably the most major, region consists of the cholinergic nuclei in the basal forebrain. The second region consists of cholinergic cells in the brainstem. The connections of the cholinergic nuclei in basal forebrain and brainstem allow widespread inuence, both directly and indirectly, on cortical and limbic system activity. The major source of cholinergic innervation of the cortex and the amygdala derives from the basal forebrain group of cells, most extensively from the nucleus basalis, and the major source of cholinergic innervation of the hippocampus derives from the medial septum and the vertical limb of the diagonal band (Aigner, 1995). Following exposure to OPs, accumulation of ACh at synapses results in rapid and profound excitotoxicity and dysfunction of cholinergic neurons in the brain due to over-activation of mAChR. Overstimulation of mAChR also disrupts the balance of glutamatergic and GABAergic activity (Lallement et al., 1991; Grasshoff et al., 2003; Santos et al., 2003) to cause neuronal excitotoxic

lesions (Wade et al., 1987; McDonough and Shih, 1997; Piekut and Phipps, 1999). The cholinergic neuronal excitotoxicity and dysfunction will lead to a loss of integrity of specic cholinergic pathways, and result in a variety of negative effects that subsequently cause neuronal cell death and neural loss in cholinergic regions of the brain that contain dense accumulations of cholinergic neurons and the majority of cholinergic projection, such as basal forebrain (piriform cortex and entorhinal cortex) and the limbic system (hippocampus, amygdala and thalamus). Seizures and respiratory depression are two common consequences of cholinergic neuronal excitotoxic lesions in the brain in the acute phase of OP poisoning. A seizure is generally observed in OP poisoned patients. In the Tokyo subway attack, approximately 3% of patients with clinical manifestations of moderate to severe sarin poisoning had convulsions (a symptom of a seizure) (Okumura et al., 1996). Seizures may result from overstimulation of central mAChRs (Savolainen and Hirvonen, 1992), and overrelease of glutamate (an excitatory neurotransmitter) from glutamatergic neurons, triggering excessive calcium release in these post-synaptic neuronal cells. Such excessive calcium release causes excitotoxic lesion to the affected neurons (McDonough and Shih, 1997). Seizures caused by cholinergic neuronal excitotoxic lesion in the brain may play a synergistic role in development of irreversible brain damage and long-term neurological and behavioral disorders (Clifford et al., 1987). However, a seizure is not essential for induction of secondary neuronal damage because it is a clinical consequence, not a cause of cholinergic neuronal excitotoxic lesion in the brain after exposure to OPs. Several studies have shown that, in absence of convulsions, neuronal cell death, neural loss, and axonal degeneration can still been found in the cortex, amygdale, hippocampus, and thalamus of monkeys exposed to soman (Hayward et al., 1990; Wall et al., 1987, 1990; Petras, 1994). This suggests that exposure to subconvulsive doses of OPs could also have long-term harmful effects on brain structure and function (Jett, 2007). Therapeutic intervention to control the convulsions is unable to halt progressive neural injury and neuronal cell death caused by OP poisoning (Bhagat et al., 2005). Respiratory depression is the leading cause of death immediately or soon after exposure to OPs. This is only seen in patients with severe OP poisoning. The depression may be independent of the respiratory muscles and secretions, and result directly from suppression of medullary respiratory-related neurons (Stewart and Anderson, 1968; Rickett et al., 1986). A recent study using anticholinergic agents to prevent respiratory arrest from dichlorvos poisoning in rats, supports the central cholinergic mechanism for respiratory depression (Bird et al., 2003). 3. Secondary neuronal damage triggered by cholinergic neuronal excitotoxicity and dysfunction Secondary neuronal damage is an indirect consequence of the initial lesion and a major contributor to the ultimate neuronal cell death and neural loss in the injured brain. Much of the damage done to the brain does not typically occur at the time of initial lesion and does not result directly from the initial lesion itself. A cascade of progressive neural injury and neuronal cell death is triggered by the initial lesion and possibly continues in the hours, days, weeks or months following the initial insult (Sullivan et al., 2000; Granacher, 2007). Secondary neuronal damage occurs due to a variety of cerebral insults including hemorrhagic stroke, ischemic stroke, traumatic brain injury, poisoning by OPs, prolonged hypoxia, infection, and neurological illness (Marion, 2003). This delayed secondary damage has come to be recognized as a major contributor to serious neuropsychiatric impairments, including memory loss, inability to concentrate, speech problems, motor and sensory decits, and behavioral problems (Beattie et al., 2000).

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Cerebral edema, inammation, calcium inux and apoptosis program have been considered as the major contributors to secondary neuronal damage in traumatic brain injury, hemorrhagic stroke, ischemic stroke, prolonged hypoxia, infection, and and Smith, 1991; Greve neurological illness (Young, 1987; Siesjo and Zink, 2009). In the rst few hours after OP poisoning, as the result of the cholinergic neuronal excitotoxicity, extensive intracellular edema (Job et al., 2007; Testylier et al., 2007; Gullapalli et al., 2010), increased BBB permeability (Carpentier et al., 1990), cerebral hemorrhages (Gokel, 2002), intracellular calcium overload (Hamilton and Posavad, 1991; Hu et al., 1991), oxidative stress (Pazdernik et al., 2001) and increased neuroinammatory and stress responses (Zimmer et al., 1998; Svensson rez et al., 2001; Williams et al., 2003; Dhote et al., 2007; Angoa-Pe et al., 2010) were generally observed in the affected brain regions. Cerebral edema is a secondary injury caused by a cascade of biochemical and pathophysiological changes triggered by the initial injury. It is closely associated with the development of secondary neuronal damage, and may be an early marker for progressive neural injury and neuronal cell death. A signicant increase in cerebral cytotoxic edema at 324 h after soman exposure, which was characterized as a decrease in apparent diffusion coefcient (ADC), was detected by the diffusionweighted magnetic resonance imaging (DW-MRI, a unique form of MRI contrast based on the translational diffusion motion of water molecules) in amygdale and hippocampus of mice (Testylier et al., 2007). Using a clinical 3.0 T MRI scanner, extensive edema that is associated with widespread neuronal death was also detected in the amygdala, piriform cortex, hippocampus, and thalamus of the guinea pigs at 67 h after exposure to soman (Gullapalli et al., 2010). Inammation is an immune response to injury, microbial invasion, or oxidative stress. The inammatory response that results from initial injury can contribute to secondary damage (Owens et al., 2001). However, if the inammatory response is excessive in the injured tissue, it can cause more severe insults than the initial injury (Tracey, 2002). Since cellular inltration in the brain in response to inammation, infection, and injury is weaker and delayed, inammatory response in the brain is induced and spread more rapidly, in contrast to other tissues (Lo et al., 2003). Inammatory cascades, such as activation of microglia, and the expression and release of classical inammatory mediators including cytokines, eicosanoids, and complement (Allan and Rothwell, 2001), stimulate the signaling networks that regulate inammatory responses to brain damage. In mice exposed to soman, a signicant increase in the mRNA levels of cytokines (IL1b, TNFa, IL-6, and SOCS3) and ICAM-1 was shown up to 7 days after OP exposure in hippocampus, cortex and cerebellum (Dhote et al., 2007). Exposure to soman caused a rapid induction of Fos (an immediate early gene protein product) and GFAP (an injuryresponse gene protein product), which is characterized by increased Fos and GFAP staining in the piriform cortex (Zimmer et al., 1998). Increased expression of neuronal COX-2 by soman was observed in the hippocampus, amygdala, piriform cortex and rez thalamus of rats from 4 h to 7 days after exposure (Angoa-Pe et al., 2010). Excessive inux of calcium due to overstimulation of mAChR and NMDA receptors is considered as an important cause of neuronal cell death following OP poisoning (Clement and Broxup, 1993; McDonough and Shih, 1997). Increased intracellular calcium accumulation was observed in cortical synaptosomes of the rats exposed to soman (Hamilton and Posavad, 1991). The altered calcium inux activates lipases, proteases, kinases, phosphatases, and endonucleases in potentially harmful metabolic cascades, thus arresting protein synthesis and depriving cells of enzymes or and Siesjo , 1996). trophic factors essential to their survival (Siesjo

In addition, intracellular calcium overload also results in free radical-related damage and apoptosis by inducing the excessive oxidative stress and inammatory responses to injury. Activation of PKC by calcium will activate the pathways involving stress and inammatory responses to injury (Pandey et al., 1999; Murasawa et al., 2000). Following exposure to OPs, apoptotic cells have been observed in different brain regions of rats (Kim et al., 1999; Kaur et al., 2007; Gunay et al., 2010). Apoptosis is a programmed cell death process that is fundamental to immune responses and is detrimental to tissue, which is often associated with secondary neuronal cell death (Lo et al., 2003). Apoptosis, characterized by nuclear DNA fragmentation, cell shrinkage, development of bubble-like blebs on the cell surface, chromatin (DNA and protein) degradation in the nucleus, and mitochondrial breakdown with the release of cytochrome C, is a prominent feature in the brain and spinal cord for as long as two months after injury (Mattson, 2000). The apoptosis program can be triggered by excessive oxygen radical formation, death-receptor (TNF or Fas receptor) ligation, DNA damage, and lysosomal protease activation. Although many signaling pathways are linked to apoptosis pathways at some level, the principal molecular components of the apoptosis program in neurons are Apaf-1, Bcl-2 proteins, the caspases, death activators (TNF, TNF receptors, Fas and FasL) and certain transcription factors (Fos, c-Jun and p53) (Yuan and Yankner, 2000). Since the mechanisms of cell proliferation, differentiation and transformation are intrinsically linked to the process of apoptosis (Romashkova and Makarov, 1999), inappropriate induction of cellular activation, aggregation, proliferation, and differentiation by the cholinergic excitotoxicity, may play a crucial role in triggering neuronal cell death. Many studies (McLeod et al., 1984; McLeod, 1985; McDonough et al., 1987; Carpentier et al., 1990, 2008; Hayward et al., 1990; Baze, 1993; Clement and Broxup, 1993; Petras, 1994; Dorandeu et al., 2005; Tetz et al., 2006; Job et al., 2007; Myhrer et al., 2007; Aroniadou-Anderjaska et al., 2009; de Araujo Furtado et al., 2009, 2010) have demonstrated that obvious neuronal cell death, neural loss and axonal degeneration were observed in different species of animals (mice, rats, cats, guinea pigs and monkeys) exposed to OPs. The piriform cortex, amygdala, hippocampus, thalamus and the cortex are the major brain regions that are predominantly affected by secondary neuronal damage. Marked histopathological alterations including edema, hemorrhage, shrunken pyknotic neurons, distal axonal degeneration, secondary demyelination and Wallerian degeneration were found 4 h to 3 months post OP-exposure in the affected brain regions. In the rats exposed to a single LD50 dose of sarin, brain damage was mainly found in the hippocampus, piriform cortex, thalamus, and amygdala. The damage was exacerbated and progressively extended to other brain regions that were not initially affected with time. At 4 h post exposure, neuronal damage was observed only in the hippocampus and the dorso-lateral thalamic nuclei; at 24 h post exposure, neuronal degeneration and edema were also observed in the piriform cortex; one week post exposure, a remarkable loss of neurons was observed in the piriform cortex, and the lesions expanded to the amygdaloid nuclei; one month post exposure, neuronal calcication was observed in both the thalamus and the piriform cortex. Hyaline plaques composed of atrophic material were found in the hippocampus; and three months post-exposure, neuronal damage was more severe than before in the hippocampus, piriform cortex, thalamus and amygdala, and the lesions expanded to the septum and the hypothalamus (Kadar et al., 1995). The irreversible progressive neuronal cell death, neural loss and axonal degeneration that are triggered by the cholinergic excitotoxicity in the brain could be the major contributors to persistent profound neurological and neuropsychiatric decits following OP poisoning. Fig. 1

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Fig. 1. Progression of damage events in the brain following exposure to OPs.

provides a simplied overview of the cascade of damage events that may occur in the brain following exposure to OPs. 4. Secondary neuronal damage and long-term neuropsychiatric and neurological disorders Considerable evidence from both animal and human studies suggests that the cholinergic nervous systems are important for learning, memory and cognition (Drachman and Leavitt, 1974; Flicker et al., 1983; Ridley et al., 1986). The inuence of cholinergic transmission upon memory and cognition could reect the higher density of cholinergic pathways within limbic and paralimbic areas. An analysis of regional variations in cholinergic innervations suggests that cortical cholinergic pathways may gate sensory information into and out of the limbic system, and that the transfer of information from sensory association cortex to limbic structures may play a pivotal role in memory and learning (Mesulam et al., 1986). Head injury, exposure to OPs, and neurodegenerative diseases such as Alzheimers disease, which involve damage to cholinergic neurons of basal forebrain and the limbic system, may cause memory, cognitive, mental, emotional, motor and sensory decits by disrupting this putative sensory-limbic gating mechanism (Mesulam, 1990; Baze, 1993; Filliat et al., 1999). Massive OP poisonings from accidental and intentional events, such as the Khamisiyah chemical munitions destruction incident and the Tokyo subway sarin attack, provided the convincing clinical data in support of long-term neurological and neuropsychiatric disorders caused by cholinergic neuronal damage and dysfunction following exposure of a civilian population to OPs. In 1991, approximately 100,000300,000 US soldiers were exposed to the OP chemical warfare agents sarin (GB) and cyclosarin (GF) when two large chemical ammunition caches were destroyed at Khamisiyah, Iraq during the Gulf War (Haley and Kurt, 1997). Studies have demonstrated that the Gulf War-deployed veterans exposed to GB/GF at Khamisiyah have been suffering from neurological and neuropsychiatric disorders (such as impaired

motor function and coordination, cognitive and memory impairments, decits in social and emotional functions, PTSD-like symptoms, etc.) (Horn et al., 1997; Proctor et al., 2006). In 1995, a Japanese cult and terrorist organization (Aum Shinrikyo) carried out the sarin gas attack at multiple locations in the Tokyo subway (Satoh and Hosokawa, 2000). Of the 5500 victims who sought emergency medical care, approximately 1000 were moderately poisoned, 50 were severely poisoned, 47 were certied as disabled, and 12 died resulted primarily from respiratory failure (Yokoyama et al., 1998). Many victims could recover from acute cholinergic symptoms (i.e. cholinergic crisis) of sarin poisoning within a couple of weeks, but mental sequelae lasted for years (Ohbu et al., 1997; Hoffman et al., 2007). Several clinical studies have shown an increased incidence of chronic neuropsychiatric disorders among the victims at different follow-up periods (6 months to 7 years after the sarin attack) (Yokoyama et al., 1998; DiGiovanni, 1999; Nishiwaki et al., 2001; Ohtani et al., 2004; Wiener and Hoffman, 2004; Miyaki et al., 2005; Gullapalli et al., 2010). Because chronic disability following brain damage is largely attributable to mental sequelae rather than focal motor or sensory neurological decits in humans (Jennett et al., 1981), neuropsychiatric disorders (such as persistent memory and cognitive impairments, psychomotor performance decits, and mental and emotional symptoms) might be the long-term prominent clinical manifestations of the victims of OP poisoning. These chronic neuropsychiatric consequences are serious public health problems caused by accidental and suicidal OP poisonings and chemical terrorist attacks, which result in the losses of many years of productive life and incur large healthcare costs. 4.1. Persistent memory and cognitive decits Memory and cognitive decits are one of the most common and persistent behavioral sequelae in victims exposed to OPs. Exposure to OP chemical warfare agents GB and GF at Khamisiyah resulted in long-term cognitive and memory impairments in the

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Gulf War-deployed veterans in 1991 (Proctor et al., 2006). Reduced hippocampal volume observed by using magnetic resonance images (MRI) in the veterans exposed to low levels of GB/GF might be closely associated with the impaired cognitive and memory functions (Chao et al., 2010). Chronic memory and cognitive impairments were also observed in the victims of the Tokyo subway sarin attack. One victim of the Tokyo subway sarin attack had severe persistent amnesia at 6 months after exposure to sarin. This patient was defective in the ability to consolidate new learning and memory (Hatta et al., 1996). The chronic decline of memory function occurred in 26 rescue team staff members and 30 police ofcers in a doseeffect manner, at 3 years after the Tokyo subway sarin attack. The rescue team staff members and police ofcers who were exposed to sarin failed to pass the backward digit span test, as compared to controls (Nishiwaki et al., 2001). 7 years after the Tokyo subway sarin attack, a chronic decline of memory function still existed in 23 subway workers exposed to sarin. The exposed subway workers performed less well in the memory function tests, and their digit number of the backward digit span test was signicantly smaller (Miyaki et al., 2005). The voxel-based morphometry revealed a signicant decrease in regional brain volumes in the hippocampus, and the insular cortex and neighboring white matter in 21 victims perceived forgetfulness with a decline of memory function 56 years after the Tokyo subway sarin attack (Yamasue et al., 2007). The hippocampus contains high levels of ACh. Neurochemical studies have reported release of ACh from the hippocampus following septal stimulation (Smith, 1974; Dudar, 1975). Taken together with the identication of high levels of the enzymes for ACh synthesis (ChAT) and degradation (AChE) in the hippocampus (Lewis and Shute, 1967; Storm-Mathisen, 1977), ACh is considered to be an important neurotransmitter in this brain region (Wainer et al., 1985). Basal forebrain including the piriform cortex is another cholinergic region of the brain. Loss of cholinergic neurons in the basal forebrain with aging results in a decline in cognitive capacity. Cholinergic neurons located in the hippocampus and basal forebrain are believed to be involved in spatial memory and memory (Scoville and Milner, 1957; Morris et al., 1982). Neuronal cell degeneration, death and loss in the hippocampus and piriform cortex resulted in serious memory and cognitive decits in animals (McDonough et al., 1986; Morita et al., 1995; Kassa et al., 2001; Bajgar et al., 2004; Myhrer et al., 2005; Joosen et al., 2009). Hippocampal and piriform cortical damage characterized by dark shrinkage degeneration of neurons, neuronal cytoplasmic lysis, nuclear pyknosis or obvious neuronal loss, might be responsible for memory and cognitive decits in dogs and rats exposed to saman (Kadar et al., 1992; Baze, 1993; Lallement et al., 1994; Kim et al., 2005; Myhrer et al., 2007; Carpentier et al., 2008; Apland et al., 2010), and in rats exposed to sarin (Kadar et al., 1995). Therefore, memory and cognitive decits in the victims of both the Khamisiyah chemical munitions destruction incident and the Tokyo subway sarin attack may be related to neuronal cell death and loss in the hippocampus and piriform cortex following OP poisoning. 4.2. Psychomotor performance decits and somatic complaints Clinical study has demonstrated that the Gulf War-deployed veterans exposed to GB/GF at Khamisiyah suffered impaired ne psychomotor dexterity, reduced visuo-spatial abilities and decits in motor function and coordination (Proctor et al., 2006). Magnetic resonance imaging studies revealed that these functional disorders might be accompanied by structural alterations in brain white matter and gray matter in the veterans exposed to the OP chemical warfare agents (Chao et al., 2010, 2011). After the Tokyo subway sarin attack, a chronic decline of psychomotor function existed in

23 subway workers exposed to sarin for 7 years. The high-exposure subway workers had a signicantly slower performance of the nger tapping tests of both the dominant and non-dominant hands than control group (Miyaki et al., 2005). In another clinical study, 29 of 38 victims of the Tokyo subway sarin attack were found to have long-lasting somatic complaints (such as gastrointestinal problems, constipation, heartburn, nausea, vomiting, colitis, migraines, headaches, back aches, and skin disorders) at 56 years after poisoning. The severity of chronic somatic complaints correlated with elevated interoceptive awareness may be related to the reduced regional white matter volume of the insular cortex identied by Voxel-based analysis of diffusion tensor MRI (Yamasue et al., 2007). Cerebral cortex and thalamus are considered to be involved in psychomotor and somatosensory functions in humans. Cerebral cortex is responsible for the planning, control, and execution of voluntary motor functions, and the thalamus is involved in relaying sensation, spatial sense and motor signals to the cerebral cortex, along with the regulation of consciousness, sleep, and alertness. Most sensory information is routed to the cerebral cortex via the thalamus. Hence, the longlasting somatic complaints and decreased psychomotor function of the victims exposed to OPs may be associated with neuronal damage in the cortex and thalamus. 4.3. Non-specic mental and emotional symptoms The Gulf War-deployed veterans who were exposed to GB/GF at Khamisiyah were found to have decits in social and emotional functions, sustained attention decit and depressive symptoms (loss of interest in daily activities, sleep changes, anger or irritability, concentration problems, etc.) in a dosedependent manner (Horn et al., 1997; Toomey et al., 2009). In the victims of the Tokyo subway sarin attack, the non-specic mental and emotional symptoms persisted for 5 years at a high rate: 65.5% of the sarin poisoning victims had recollections of an event; 48.3% of the victims had avoidance of places that trigger recollections of the trauma; 43.3% had tension and 42.9% had forgetfulness (Ohtani et al., 2004). Past study showed that increasing anxiety, psychomotor depression, intellectual impairment, and unusual dreams were also observed in the sarinpoisoned victims (Bowers et al., 1964). Unfortunately, the mechanisms underlying non-specic mental and emotional disorders are largely unknown. Secondary neuronal damage in some cholinergic regions of the brain (such as the cortex, amygdala, hippocampus and thalamus) may involve the development of these neuropsychiatric disorders. 4.4. Potential secondary damage effects 4.4.1. Posttraumatic stress disorder (PTSD) PTSD symptoms were observed for up to 6 years in the victims of the Tokyo subway sarin attack. The patients who were diagnosed with PTSD had anxiety, fear, nightmares, insomnia, irritability, ashbacks and depression from months to years (Ohbu et al., 1997). Their PTSD symptoms were often associated with other neuropsychiatric symptoms (fatigue, tension-anxiety, depression, and angerhostility) and psychomotor performance (i.e. motor persistence, sustained attention, response speed, and visuomotor coordination) (Yokoyama et al., 1998). Structural abnormalities of the left anterior cingulate cortex (ACC) gray matter was found by using voxel-based morphometry in 36 victims with PTSD 56 years after sarin poisoning. The results suggest that PTSD may be associated with a hyper-responsive amygdala, and an under-responsive ACC that fails to inhibit the amygdale (Yamasue et al., 2003). In addition, smaller regional volumes in the right insular cortex, right temporal cortex, left hippocampus, and left temporal stem were identied by

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Fig. 2. Potential neuroprotective strategies targeting each of the four phases of OP-induced brain damage.

Voxel-based analysis of diffusion tensor MRI in 9 victims with PTSD (Yamasue et al., 2007). PTSD is closely related to brain damage such as traumatic brain injury (TBI). Many PTSD symptoms (depression, anxiety, memory and attention decits, sleep problems and emotional disturbances) overlap with symptoms of TBI (King, 2008; Kennedy et al., 2010). Clinical studies indicate that more than 80% of patients with TBI develop chronic PTSD (Bryant and Harvey, 1998; Mayou et al., 2000; Bryant, 2001). PTSD is more likely to be a psychological consequence of brain damage and may result from secondary neuronal damage after brain injury (Chen and Huang, 2011). A recent study has found a causal link between TBI and PTSD. The rats suffering from concussive brain trauma appeared to overgeneralize learned fear to both conditioned and novel stimuli. A signicant upregulation of excitatory N-methyl-D-aspartate receptors was observed in the basolateral amygdala complex, suggesting that amygdaloid neurons are easily excited by brain trauma to acquire inappropriately strong fear, and the damaged amygdala increases susceptibility to PTSD (Reger et al., 2012). The amygdala and the hippocampus, the two important structures of the emotional memory system in the brain, have been conrmed to be involved in PTSD (Pitman et al., 2001). Secondary neuronal damage (neuronal cell death, neural loss and axonal degeneration) caused by OP poisoning in the amygdala and the hippocampus (McLeod, 1985; McDonough et al., 1986; Baze, 1993; Petras, 1994; Morita et al., 1995; Myhrer et al., 2007; Aroniadou-Anderjaska et al., 2009; Apland et al., 2010), may increase vulnerability to PTSD. However, further experimental and clinical research will be needed to explore the link between OP-induced brain damage and PTSD. 4.4.2. Brain cancer A 10-year follow-up study has found an increased risk (an approximately twofold excess) of brain cancer death among the Gulf War-deployed veterans exposed to GB and GF at Khamisiyah, Iraq in March 1991. The risk of brain cancer death, which increased with the time and amount of exposure, is believed to develop over 1020 years in the veterans exposed to sarin (Bullman et al., 2005). OP-induced brain damage may increase risk of brain cancer in the victims exposed to OPs. 5. Potential therapeutic strategies for victims of OP poisoning OP-induced brain damage, to date, is an irreversible neuronal injury (Deshpande et al., 2010), because, no pharmacological treatment is currently available to prevent or block secondary damage processes (Scalea, 2005). However, secondary neuronal damage processes offer a potential therapeutic window of

opportunity (Armin et al., 2006) in which progressive neural injury and neuronal cell death may be prevented and the extent of disability may be reduced during the rst few hours (Marion, 2003) after exposure to OPs. Therapeutic strategies aimed at limiting or preventing secondary neuronal damage at the early stage of injury (within hours after exposure), may halt or mitigate progressive neuronal cell death and degeneration that are caused by the cholinergic neuronal excitotoxic events such as edema, inammation, ionic imbalance, and apoptosis. It may reduce serious neuropsychiatric impairments and help improve recovery of neurological and neuropsychiatric functions after exposure to OPs, thus leading to a signicant decrease in both the direct cost for medical care and indirect cost of lost productivity resulting from OP-induced brain damage. Based on the mechanism of action of OPs and secondary neuronal damage processes triggered by OP poisoning in the brain, the development of OP-induced brain damage can be divided into four phases: OP toxicity phase, cholinergic excitotoxicity phase, secondary neuronal damage phase, and chronic symptom development phase. Potential neuroprotective strategies targeting each of the four phases of OP-induced brain damage are described in Fig. 2. 5.1. Antidote therapies in the OP toxicity phase The OP toxicity phase starts from OP poisoning until induction of cholinergic neuronal excitotoxicity in the brain, which takes about 230 min, depending on the amount of exposure. Antidotes such as atropine and related anticholinergic drugs may be the effective against OP poisoning in this phase (Singh et al., 1995; Kventsel et al., 2005; Eyer et al., 2009; Thiermann et al., 2011). However, some anticholinergic drugs (such as dicyclomine, benactyzine, scopolamine, etc.) have been reported to cause a range of anticholinergic side effects (such as dry mouth and nausea, dizziness, drowsiness, blurred vision and drowsiness), and increase the risk of cognitive impairments and dementia (Campbell et al., 2010). Consequently, they are no longer widely used in clinical practice. Novel, high efcient and low-toxic anticholinergic drugs eagerly need to be discovered and further developed for clinical use. In addition, there is a very limited therapeutic time window for antidotes. Antidotes may not have a neuroprotective effect against OP poisoning in the brain once cholinergic neuronal excitotoxicity is induced after 30 min post exposure. Oxime compounds, which are considered to be used as antidotes for OPs (Kassa, 2002; Bajgar et al., 2007; Masson, 2011), may reactivate AChE by attaching to the phosphorus atom and forming an oxime-phosphonate which then splits away from the AChE

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molecule. Unfortunately, most oxime compounds including the oximes that can pass through the bloodbrain barrier have been observed to fail in bringing AChE back to normal level. MINA (monoisonitrosoacetone), one of oxime compounds, has been conrmed to penetrate the bloodbrain barrier (BBB) easily (Garcia et al., 2010). MINA at the highest dose of 139.3 mg/kg reactivated only 3146% of AChE activity inhibited by GB in peripheral tissues (heart, diaphragm and skeletal muscle) of guinea pigs at 60 minutes after exposure to GB. However, it also just reactivated 3144% of AChE activity inhibited by GB in the brain tissues (brainstem, cerebellum, cortex, hippocampus, midbrain and striatum) (Skovira et al., 2010). Hence, the BBB may not be the main barrier to effective treatment with oximes for OP poisoning. An effective oxime compound for antidotal treatment should not only pass through the BBB but also be able to achieve the maximum reactivation (e.g. more than 50%) of AChE activity inhibited by OPs in the brain. A short therapeutic time window (within 30 min after exposure to OPs) could be also crucial for the oxime compound to be effective against OPs. 5.2. Anti-excitotoxic therapies in the cholinergic excitotoxicity phase The cholinergic excitotoxicity phase begins with induction of cholinergic neuronal excitotoxicity and ends until secondary neuronal damage occurs, which takes approximately 16 h. In this phase, neuroprotective strategies may target on reducing cerebral edema and inammatory reaction, attenuating excitotoxicity by blocking calcium inux, and inhibiting apoptosis program. Osmotherapy using mannitol or diuretics is a good treatment approach for cerebral edema (Hays et al., 2011). Dihydropyridine calcium channel blockers (such as Amlodipine, Azelnidipine, Benidipine, Isradipine, Nicardipine, Pranidipine, etc.) (van der Stelt et al., 2002), and humanin peptides (including HN, HNG and other mutants) (Zou et al., 2003) may be effective in attenuating excitotoxicity by blocking calcium inux. Steroidal and non-steroidal anti-inammatory drugs may be used to reduce inammation or swelling in the brain (Hoozemans et al., 2003; Gomes et al., 2005) following OP exposure. Treatments aimed to inhibiting apoptosis may focus on blocking the expression of pro-apoptotic factors and promoting the expression of anti-apoptotic factors (Fabregat, 2009). FLICE-inhibitory proteins, Bcl-2, CrmA, IAPs, and the Akt protein kinase may be effective in preventing apoptosis program (Uren et al., 1996; Rane and Klein, 2009; Lindsay et al., 2011; Plati et al., 2011). In addition, benzodiazepine treatment may help attenuating seizures, muscle spasms, anxiety, insomnia, agitation, and other cholinergic excitotoxic symptoms in this phase. Benzodiazepines have sedative, hypnotic, anticonvulsant, myorelaxant, anxiolytic, and amnesic properties by increasing the effects of GABA in the brain (Sand et al., 2000). 5.3. Neuroprotective strategies against secondary neuronal damage in the secondary neuronal damage phase The secondary neuronal damage phase is marked by which neuronal cell death, neural loss and axonal degeneration occur and continue developing in the cholinergic regions of the brain affected by OPs. This phase may start from 6 h after OP exposure and may last several months. The only measure of treatment in this phase is to slow down neuronal cell death, neural loss and axonal degeneration. Antioxidants and NMDAR antagonists have been reported to protect against oxidative injury and neurodegeneration caused by OP in rats brain. Pretreatment with antioxidant (vitamin E or PBN) or a NMDA receptor antagonist (memantine) can suppress OP-induced oxidative/nitrosative damage and dendritic neurodegeneration of pyramidal neurons

in the hippocampal CA1 eld of the rats at 1 h after exposure to an OP compound, DFP (Zaja-Milatovic et al., 2009). Oral-administration of mitochondria-targeted antioxidant MitoQ reduced oxidative damage, mitochondrial dysfunction and cell death induced by OP (dichlorvos) exposure in rat brain. After treatment with MitoQ, a signicant decrease in oxidative stress parameters (mitochondrial ROS production, lipid peroxidation, protein and DNA oxidation, DNA fragmentation, cytochrome C release and caspase-3 activity) was found to be accompanied with the reduced mitochondrial swelling and restoration of normal mitochondrial morphology and function in the rats exposed to dichlorvos (Wani et al., 2011). Antioxidant therapy may be a potential pharmacological approach to prevent apoptotic cell death and to slow down secondary neuronal damage processes in the brain following OP poisoning. Some cytokines (GDNF, FGF2 and IFN) have previously been reported to have the ability to slow down neuronal cell death and degeneration (Onyango et al., 2005; Jin et al., 2007; Zucchini et al., 2008). Recently, a cytokine treatment (cocktail of EGF and FGF-2) that attempted to promote neurogenesis (a natural regenerative process) in the brain of mice exposed to soman has been reported. Interestingly, the combination treatment of EGF and FGF-2 enhanced neuronal regeneration in the hippocampal CA1 eld of soman-poisoned mice. The enhanced neuronal regeneration boosted by the cytokine treatment might contribute to partial recovery of neurobehavioral functions in mice following exposure to soman (Collombet, 2011). The new therapeutical strategy that combines two or more cytokines (interleukins, chemokines, growth factors, neurotrophins, lymphokines, monokines, interferons, colony-stimulating factors, immunomodulators, and immunotransmitters) may have the potential to halt or mitigate secondary neuronal damage inicted by OP poisoning in this phase. Collombet (2011) also reported a cell therapy in which BrdU or Qtracker nanocrystal-labeled human mesenchymal stem cells (bone marrow stromal cells) were injected intracerebrally by stereotaxic procedure into the hippocampus of soman-poisoned mice. However, after intracerebral injection of mesenchymal stem cell, engrafted stem cells were observed only in the injection site of the hippocampus of soman-poisoned mice on post-soman days 21. There was no migration of grafted stem cells to be found in other brain regions. It should be noted that stem cell therapy is still in the experimental stages. The in vivo stem cell microenvironment that regulates stem cell characteristics within the niche in the human body is largely unknown. The proper microenvironment, including cellcell interactions between stem cells, interactions between stem cells and neighboring differentiated cells, interactions between stem cells and adhesion molecules, extracellular matrix components, the oxygen tension, growth factors, cytokines, the pH, ionic strength, and ATP metabolites, etc., is very important to promote stem cell proliferation and differentiation into the right cellular phenotype and to form the right tissue. In absence of the proper microenvironment, the simple location of stem cells in a tissue or organ is not able to induce stem cells to proliferate and differentiate into the right type of cells. Sometimes, it may cause stem cells to differentiate into undesired cells (such as tumor cells) (Jotzu et al., 2010). In addition, unlike traumatic brain injury or ischemic stroke that causes an obvious focal zone of necrosis/infarction or a necrotic cavity in the brain, OP-induced brain damage is characterized by diffuse neuronal cell death, neural loss and axonal degeneration in different brain regions (the piriform cortex, amygdala, hippocampus, thalamus and the cortex). Stem cell therapy may not be effective for OP-induced brain damage because it is impossible to transplant stem cells to all the cholinergic regions of the brain affected by OPs.

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Y. Chen / NeuroToxicology 33 (2012) 391400 Allan SM, Rothwell NJ. Cytokines and acute neurodegeneration. Nat Rev Neurosci 2001;2:73444. rez M, Kreipke CW, Thomas DM, Van Shura KE, Lyman M, McDonough JH, et Angoa-Pe al. Soman increases neuronal COX-2 levels: possible link between seizures and protracted neuronal damage. Neurotoxicology 2010;31:73846. Apland JP, Figueiredo TH, Qashu F, Aroniadou-Anderjaska V, Souza AP, Braga MF. Higher susceptibility of the ventral versus the dorsal hippocampus and the posteroventral versus anterodorsal amygdala to soman-induced neuropathology. Neurotoxicology 2010;31:48592. Armin SS, Colohan AR, Zhang JH. Traumatic subarachnoid hemorrhage: our current understanding and its evolution over the past half century. Neurol Res 2006;28:44552. Aroniadou-Anderjaska V, Figueiredo TH, Apland JP, Qashu F, Braga MF. Primary brain targets of nerve agents: the role of the amygdala in comparison to the hippocampus. Neurotoxicology 2009;30:7726. 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5.4. Therapies targeting individual symptoms or clusters in the development phase of chronic neuropsychiatric and neurological symptoms The development phase of chronic neuropsychiatric and neurological symptoms is the longest phase in the development of OPinduced brain damage. In this phase, most of chronic neuropsychiatric and neurological symptoms may start approximately in a couple months after OP exposure and persist for years. Currently, there is no effective treatment against chronic neuropsychiatric and neurological disorders caused by OP poisoning. Pharmacotherapy aimed at targeting individual symptoms or clusters of chronic neuropsychiatric and neurological disorders (De Wester, 1996; Sink et al., 2005; Ravindran and Stein, 2010) may help improve recovery of neurobehavioral functions in the victims of OP poisoning. 6. Conclusions Considerable laboratory evidence from animal studies have showed that progressive neuronal cell death, neural loss and axonal degeneration occurred after OP exposure in the cholinergic regions of the brain that are predominantly affected by toxicity of OPs. This delayed secondary neuronal damage could result from cholinergic neuronal excitotoxicity and dysfunction that are caused by OP-induced irreversible AChE inhibition in the brain. It may largely involve the development of persistent profound neuropsychiatric and neurological decits (such as cognitive and memory impairments, psychomotor performance decits, somatic complaints, non-specic mental and emotional symptoms, etc.) following OP poisoning. Antidotes including atropine and related anticholinergic drugs may not be sufcient to prevent the cholinergic neuronal excitotoxicity and to block secondary neuronal damage, because there is a very limited therapeutic time window (within 30 min) for current antidotes. Prompt therapies that target attenuation of cholinergic excitotoxicity may halt or mitigate secondary neuronal damage. Therapeutic strategies to reduce cholinergic excitotoxicity should include reducing cerebral edema and inammatory reaction, blocking calcium inux, inhibiting apoptosis program, and controlling seizures. However, therapeutic intervention to control the convulsions may not be able to halt secondary neuronal damage triggered by the cholinergic neuronal excitotoxicity, because a seizure is only a clinical consequence, not a cause of cholinergic neuronal excitotoxic lesion in the brain. Neuroprotective strategies for slowing down the process of secondary neuronal damage may help attenuate neurological and neuropsychiatric decits and improve neurobehavioral functions in the OP-exposed patients. Therapeutic intervention with cytokines, antioxidants and NMDAR antagonists (either single-agent therapy or combination therapy) may have the potential to prevent apoptotic cell death and to slow down secondary neuronal damage processes in the brain. Conict of interest statement The authors report no declarations of interest. Acknowledgments The author would like to thank Mr. Jeffrey A. Koenig and Mr. Haoxing Chen for their valuable assistance in the preparation of this manuscript. References
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