Gerald P. H. Ballough

Microtubule-associated protein 2 (MAP-2): a sensitive marker of seizure-related brain damage.

We have assessed the efficacy of MAP-2 immunohistochemistry as a marker of seizure-related brain damage and its suitability for quantitation of the damage using densitometric and morphometric image analysis. Seizures were produced in rats by administration of 1.5 LD50 soman, an irreversible AChE inhibitor. Our results demonstrate that neuronal damage, assessed using hematoxylin and eosin, and cresyl violet staining, was colocalized on adjacent serial sections with clearly demarcated reductions in MAP-2 staining. The most severely damaged brain regions were devoid of MAP-2 staining. Reductions in MAP-2 immunostaining were found to be exceptionally well suited for quantitation using densitometric and morphometric image analysis. This study represents the first demonstration of seizure-induced excitotoxic alterations in MAP-2.

Neuroprotective Effects of HU‐211 on Brain Damage Resulting from Soman‐Induced Seizures

ABSTRACT: Neuroprotective effects of HU‐211 (dexanabinol), a synthetic nonpsychotropic analog of tetrahydrocannabinol, on brain damage resulting from soman‐induced seizures were examined in male Sprague‐Dawley rats challenged with 1.6 LD50 soman. At 5 or 40 min after onset of seizures, the rats were given an intraperitoneal injection of 25 mg/kg HU‐211. All rats that received soman showed electrocorticographic (ECoG) evidence of sustained seizures and status epilepticus for 4–6 hr. HU‐211 had no effect on either the strength or duration of seizure activity. Administration of HU‐211 at 5 min after seizure onset reduced median lesion volume 86% (as assessed by microtubule‐associated protein 2 (MAP2)‐negative staining), and when administered 40 min post‐onset, the reduction in necrosis was 81.5% despite the presence of continuous seizures for 4–5 hr. These observations were corroborated by hemotoxylin and eosin (H&E) histopathological assessment that showed a significant reduction in piriform cortical neuronal damage in HU‐211‐treated animals. It is concluded that HU‐211 provides considerable neuroprotection against brain damage produced by soman‐induced seizures.

GM1 monosialoganglioside pretreatment protects against soman-induced seizure-related brain damage

The effects of GM1 monosialoganglioside pretreatment on brain damage resulting from soman-induced seizure activity were examined in this study. Male Sprague-Dawley rats were infused with GM1 via an osmotic minipump connected through a permanent cannula implanted intracerebroventricularly and challenged with soman (83 micrograms/kg, i.e., 1.25 x LD50) 4 d after initiation of GM1 infusion. Electrocorticographic recordings were monitored via indwelling cortical electrodes. Twenty-seven hours after soman administration, anesthetized rats were euthanized via transcardial perfusion with buffered paraformaldehyde. Brains were processed for hematoxylin and eosin (H&E), cresyl violet (CV), and acetylcholinesterase (AChE) histochemistry, and glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2) immunohistochemistry. All soman-challenged rats not infused with GM1 (n = 14) developed status epilepticus (SE).

Benzamide, a Poly(ADP-Ribose) Polymerase Inhibitor, Is Neuroprotective against Soman-Induced Seizure-Related Brain Damagea

Soman is an organophosphorous nerve agent that irreversibly inhibits acetylcholinesterase, causing a rapid rise in acetylcholine levels. This high elevation of acetylcholine over a 5–20-min period leads to limbic seizures and seizure-related brain damage (SRBD).1 There is considerable evidence that soman-induced SRBD stems from glutamate excitotoxicity,2 which, in turn, generates DNA-damaging free radicals. This hypothesis is supported by the exacerbation in excitotoxicity of glutamate due to a decrease in adenosine triphosphate (ATP).3,4 The linkage of DNA damage, metabolic impairment, and cell death was first formulated by Berger.5 He formulated an energy-dependent hypothesis to explain the cytotoxic action of DNA alkylating agents. He proposed that alkylation of DNA causes activation of the nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30, PARP) and results in depletion of ATP. This hypothesis appears to be relevant to any cytotoxic event in which DNA damage causes depletion of cellular ATP. Bis-(2-chloroethyl) sulfide (SM), a potent DNA alkylating agent, was used to study and validate Berger’s hypothesis. The mechanism of SM cytotoxicity in human lymphocytes was shown to result in necrotic cell death,6 which is a consequence of decreasing ATP.7 SM also causes disruption of the nuclear and plasma membranes8 as well as other biochemical and morphologic changes in the lymphocytes consistent with necrotic cell death.9 Inhibitors of PARP (PARPI) were effective at altering or blocking the SM-dependent changes.6,7,9 PARPI converts the SM-initiated necrotic pattern of cell death to what appears to be noninflammatory apoptotic changes.6 Since both soman and SM have been shown to cause energy-dependent necrotic cell death, it was decided to examine whether PARPI would be as effective at blocking the cellular damage of SRBD as the PARPI were in reducing SM-induced cellular injury. This decision was supported by the findings that the prevention of ATP depletion by administration of PARPI provides considerable neuroprotection in various excitotoxic models.11,12

Effects of soman-induced convulsions on phosphoinositide metabolism

Turnover of [3H]phosphoinositides (PI) was examined in brain slices from the hippocampus of rats undergoing soman-induced seizure activity. Hydrolysis of PI was determined by measuring the accumulation of [3H]inositol-1-phosphate (IP1). Incubation of hippocampal slices in the presence of carbachol or norepinephrine (NE) increased PI hydrolysis. Stimulated hydrolysis by NE, but not carbachol was significantly reduced in slices from soman-challenged rats undergoing convulsive activity. NE-stimulated PI hydrolysis was not reduced in slices from animals exposed to soman that did not exhibit convulsive activity. In rats surviving for 24 h, the response to NE was not different from control rats. In control slices, NE-stimulated hydrolysis of PI was potentiated by GABA. No potentiation by GABA was seen in slices from animals undergoing seizures. Uptake and incorporation of myo-[2-3H]inositol into phospholipids was reduced in slices from rats undergoing convulsions. Reduced IP1 production appeared to be owing, in part, to decreased synthesis of inositol lipids. These observations suggest that during soman-induced seizure activity, there is an apparent decrease in the response of the PI second messenger system to NE stimulation, and that this may contribute to the severity and duration of convulsions and brain damage resulting from exposure to soman and other anticholinesterase compounds.

RESIDUAL ACETYLCHOLINESTERASE ACTIVITY IN THE BASAL LATERAL AMYGDALA FOLLOWING SOMAN-INDUCED STATUS EPILEPTICUS

Soman (pinacolylmethylphosphonofluoridate) causes seizures and seizurerelated brain damageresulting from theirreversibleinhibition of acetylcholinesterase (AChE) in the central nervous system (CNS). The authors have examined the possibility that the loss of AChE reactivity in discrete brain regions may have importance in the development of status epilepticus caused by soman. In these studies, male Sprague-Dawley rats were given GM monosialoganglio1 side, intracerebroventricularly, via an osmotic minipump. Four days after initiation of GM1 infusions, rats were injected with 1.25 LD50 soman (83mug/kg,im). Electrocorticogram (ECoG) recordings were monitored via indwelling cortical electrodes. All rats were euthanized 27 h after soman administration. Brain tissue was sucrose-saturated, cryostat-sectioned, and processed for AChE histochemistry. A Quantimet 600 Image Analysis System and Biological Microscope were used to assess the optical density of histochemical staining for AChE in the piriform cortex, basol...