Soman-induced convulsions: The neuropathology revisited
Introduction
Organophosphates (OP) such as soman (O-1,2,2-trimethylpropylmethyl-phosphono-fluoridate), a chemical warfare agent, act by inhibiting mainly acetylcholinesterase (AChE) both peripherally and centrally. Acute exposure to soman can cause profound physiological debilitation, generalized seizures, coma and death (Shih and Scremin, 1992, McDonough and Shih, 1997, Brown and Brix, 1998). In survivors, the development of long-lasting seizure activity is known to be strongly related to the occurrence of brain damage (Petras, 1981, Lemercier et al., 1983, McLeod et al., 1984, McDonough and Shih, 1997, Carpentier et al., 2000). Neurochemically, acetylcholine accumulation plays a key-role in the initiation of seizure activity whereas the excitatory amino acid glutamate is involved in the long-term maintenance of epileptiform discharges and also in the causation of neuronal death (Lallement et al., 1991a, Lallement et al., 1991b, Lallement et al., 1991c, Lallement et al., 1992a, Lallement et al., 1992b, Lallement et al., 1993, McDonough and Shih, 1997, Shih and McDonough, 1997). Thus, in soman poisoning, as in other seizure-related neuropathology, the neuronal death is largely due to the overactivation of certain glutamate receptors, and can be classified as “excitotoxic”. This latter assertion is sustained by numerous physiological, biochemical and pharmacological evidences listed in the review of McDonough and Shih (1997). Indeed, the possibility that ischaemic/anoxic/hypoxic mechanisms might participate in soman-induced brain damage have been often mentioned. However, no clear-cut evidences favor this hypothesis (e.g. McDonough and Shih, 1997 and references therein). Anyway, whatever the actual initial cause would be, the detailed mechanisms of cell disintegration have not yet been elucidated.
The terms apoptosis and necrosis, the most widely recognized forms of cell death, were originally introduced for describing distinct morphological changes at the ultrastructural level (Kerr et al., 1972, Searle et al., 1982, Walker et al., 1988). Thus, apoptotic cells are shrunken and condensed, and early in the cell death process they have pycnotic nuclei displaying large, crescent-shaped or round chromatin clumps, whereas the cytoplasmic organelles and plasma membranes remain intact until later stages of degeneration. In contrast, necrotic cells are swollen with nuclei showing small, irregular and dispersed clumps of chromatin, and at an early stage the cytoplasm is vacuolated, containing swollen reticulum and mitochondria, and the plasma membrane may be disrupted. With these simple dichotomic ultrastructural descriptions, it seems rather easy to separate apoptosis from necrosis. But, in fact, the morphology can be highly variable and relatively difficult to assess as a defining characteristic of a particular kind of cell death. Thus, it seems that apoptosis, necrosis and hybrid forms can coexist and a possible continuum from apoptosis to necrosis has even been suggested (Leist and Nicotera, 1997; Portera Cailliau et al., 1997a, Portera Cailliau et al., 1997b, Raffray and Cohen, 1997, Leist and Nicotera, 1998, Martin et al., 1998, Roy and Sapolsky, 1999, MacManus and Buchan, 2000, Saraste and Pulkki, 2000, Graham and Chen, 2001, Moroni et al., 2001). Additional types of cell death such as, necrapoptosis, parapoptosis and autophagic cell death have also been described (Clarke, 1990, Lemasters, 1999, Sperandio et al., 2000) and other classifications have been proposed (Clarke, 1990, Olney and Ishimaru, 1999).
Another difficulty comes from the confusion surrounding the definitions of apoptosis and programmed cell death (PCD). Since the morphological features that originally served to define apoptosis were suggestive of an “active” process of cell death, the concept then arose that apoptosis might be a physiological process of cell death for regulating cell number by counterbalancing mitosis, although it was held that the phenomenon could also be triggered in some pathological situations. PCD refers to any “active” process that requires intracellular mechanisms for cell death to occur and is also associated with “suicidal” circumstances (cell death during development, homeostatic regulation of cell numbers, elimination of cancer cells and of self directed immune system cells, etc.) in which cells can be forced to activate death genes and death programs. However, in contrast to apoptosis, the concept of PCD is exclusively mechanistic in nature and independent of any morphological features. Historically, the identification of a PCD process was based on the detection of DNA changes through in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL technique; Gavrieli et al., 1992) and internucleosomal DNA cleavage (DNA laddering) as well as on the detection of certain biochemical markers associated with the activation of programmed cell death mechanisms. The latter include mRNA and protein expression of multiple genes such as p53, Bax, cyclin D1, and the activation of caspases. Unfortunately, the link between the concepts of apoptosis and of PCD had led some to regard the two terms as synonymous and opposed to the concept of “necrosis” which was originally believed to be a “passive” cell death in which cellular processes played no role. Consequently, regardless of the morphological criteria, “active” cell death of all types was frequently called “apoptosis”, and the mere presence of nuclear condensation or of biochemical changes normally associated with PCD were considered adequate criteria for labeling dying cells as “apoptotic”. However, recent studies (e.g. Fujikawa, 2000, Fujikawa et al., 2000a, Fujikawa et al., 2000b) have shown that PCD mechanisms (evidenced by biochemical markers) can contribute to necrotic as well as to apoptotic neuronal death (assessed by ultrastructural examination), and the nonrigorous reliance on PCD criteria alone, has led in many cases to the misidentification of nonapoptotic neuronal death after ischemia, seizures, cerebral trauma and other neurological insults as apoptosis. Confusion about the definition of apoptosis, which is still a matter of controversy (Fujikawa, 2000, Sloviter, 2002), might be not only a formal problem of terminology but also a potential source of serious therapeutical errors. Thus, as stated by Sloviter (2002), if apoptosis is a physiological process of cell elimination, anti-apoptotic drugs might be double-edged swords that could cause cancer, autoimmune disorders, chronic inflammation and development abnormalities.
In the specific field of soman poisoning, several ultrastructural studies of neuronal death were performed in the 1980s, but none of them dealt with the notion of apoptosis (Sikora-VanMeter et al., 1985, Wall et al., 1985, McLeod and Wall, 1987, Sikora-VanMeter et al., 1987, Lebeda et al., 1988). Recently, the possible involvement of apoptosis after soman intoxication was proposed (Filbert and Ballough, 1997, Ballough et al., 1998), but this hypothesis was based mainly on p53 expression and TUNEL labeling. In one work, the authors stated that “the presence of apoptotic cells was confirmed by transmission electron microscopy” (Ballough et al., 1998). Unfortunately, the results of these investigations are only available in the form of poster abstracts and have not been published in detail. Current data about soman-induced apoptosis appears thus rather incomplete. In spite of this, there is at the present time an awkward tendency to qualify soman-induced neuronal death as “apoptotic” without proper ultrastructural evidence.
We have therefore re-investigated the pathogenesis of soman-induced brain damage throughout the first 24 h after intoxication by a convulsant dose of soman, combining a descriptive morphopathological study at light and electron microscopy levels with a concomitant investigation of several PCD markers, including the expression of p53 and Bax proteins and the in situ detection of DNA fragmentation by TUNEL labeling. As complementary studies, cell death, tissue healing and glial reactions were also examined 7 days after the poisoning. With regard to soman-induced brain damage, such a multiparametric study has never before been performed.
Section snippets
Drugs
Soman, >97% pure by chromatography, was supplied by the Centre d’Etudes du Bouchet (Vert Le Petit, France). It was freshly prepared by diluting the initial concentrated solution (2 mg/ml in isopropanol) in ice-cold 0.9% (w/v) saline.
The oxime HI-6 dichloride, a peripheral reactivator of cholinesterase, which is effective in prolonging the survival time of intoxicated animals without affecting convulsions, was a generous gift of DRDC (Suffield, Canada).
Animals and experimental procedure
Adult male Swiss mice (30 g; Janvier, France)
Clinical response to soman poisoning
Within 10 min, systemic administration of soman 172 μg/kg produced various symptoms including chewing, hypersalivation, fasciculation, tremors and respiratory distress which sometimes led to death (9 out of the 55 mice which were planned to be sacrificed later, died within the first hour after the intoxication). Above all, most of the intoxicated mice experienced tonic–clonic convulsions, which generally lasted for hours (Table 1). As expected in this model of intoxication, convulsion-free mice
Discussion
As a first main result, the present work fully confirms that occurrence of soman-induced brain damage depends on the development of convulsions and that the topographically less extensive and less severe cellular damage depends on the duration of convulsions. In addition, we found that, among the various types or stages of cell death produced by a convulsive dose of soman, those which closely resemble the ultrastructural morphology of an “apoptotic” dying cell were rather rare. Our results also
Conclusions
In conclusion, the present study confirms the relationship that exists between soman-induced convulsions and neuropathology. Thus, even a short sequence of convulsions (<2 h) was shown to produce detectable cell damage within the first 24 h of the intoxication. In the case of long-lasting convulsions, cell death processes, axonal degeneration and intense glial reactivity were still ongoing 7 days after the intoxication. Above all, the present study is the first, to our knowledge, to investigate
Acknowledgments
This work was supported by grants from the Direction Générale de l’Armement (DGA; Contract number 98 10064) and from the Direction Centrale du Service de Santé des Armées (DCSSA). The authors would consider thanking Mr. S. Mure for his expert assistance with the microphotographs.
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2021, Toxicology LettersCitation Excerpt :Understanding the mechanism of damage is critical for developing improved treatments. Certainly, one contributor to neuronal injury is generalized tonic-clonic seizures (McDonough et al., 1987; Petras, 1994) that can progress into status epilepticus (Baille et al., 2005; Lallement et al., 1993). The overstimulation of glutamatergic neurons results in excessive glutamate release and excitotoxicity (Lallement et al., 1991).