Cerebral edema induced in mice by a convulsive dose of soman. Evaluation through diffusion-weighted magnetic resonance imaging and histology.

PURPOSE: In the present study, diffusion-weighted magnetic resonance imaging (DW-MRI) and histology were used to assess cerebral edema and lesions in mice intoxicated by a convulsive dose of soman, an organophosphate compound acting as an irreversible cholinesterase inhibitor. METHODS: Three hours and 24 h after the intoxication with soman (172 microg/kg), the mice were anesthetized with an isoflurane/N(2)O mixture and their brain examined with DW-MRI. After the imaging sessions, the mice were sacrificed for histological analysis of their brain. RESULTS: A decrease in the apparent diffusion coefficient (ADC) was detected as soon as 3 h after the intoxication and was found strongly enhanced at 24 h. A correlation was obtained between the ADC change and the severity of the overall brain damage (edema and cellular degeneration): the more severe the damage, the stronger the ADC drop. Anesthesia was shown to interrupt soman-induced seizures and to attenuate edema and cell change in certain sensitive brain areas. Finally, brain water content was assessed using the traditional dry/wet weight method. A significant increase of brain water was observed following the intoxication. CONCLUSIONS: The ADC decrease observed in the present study suggests that brain edema in soman poisoning is mainly intracellular and cytotoxic. Since entry of water into the brain was also evidenced, this type of edema is certainly mixed with others (vasogenic, hydrostatic, osmotic). The present study confirms the potential of DW-MRI as a non-invasive tool for monitoring the acute neuropathological consequences (edema and neurodegeneration) of soman-induced seizures.

Soman poisoning induces delayed astrogliotic scar and angiogenesis in damaged mouse brain areas.

Gliotic scar formation and angiogenesis are two biological events involved in the tissue reparative process generally occurring in the brain after mechanically induced injury, ischemia or cerebral tumor development. For the first time, in this study, neo-vascularization and glial scar formation were investigated in the brain of soman-poisoned mice over a 3-month period after nerve agent exposure (1.2 LD50 of soman). Using anti-claudin-5 and anti-vascular endothelial growth factor (VEGF) immunostaining techniques on brain sections, blood vessels were quantified and VEGF expression was verified to appraise the level of neo-angiogenesis induced in damaged brain areas. Furthermore, glial scar formation and neuropathology were estimated over time in the same injured brain regions by anti-glial fibrillary acidic protein (GFAP) immunohistochemistry and hemalun-phloxin (H&P) dye staining, respectively. VEGF over-expression was noticed on post-soman day 3 in lesioned areas such as the hippocampal CA1 field and amygdala. This was followed by an increase in the quantity of mature blood vessels, 3 months after soman poisoning, in the same brain areas. On the other hand, massive astroglial cell activation was demonstrated on post-soman day 8. Reactive astroglial cells were located only in damaged cerebral regions where H&P-stained eosinophilic neurons were found. For longer experimental times, astroglial response slowly decreased overtime but remained detectable on post-soman day 90 in some discrete brain regions (i.e. CA1 field and amygdala) evidencing the formation of a glial scar. In this study, we discuss the key role of VEGF in the angiogenic process and in the glial or neuronal response induced by soman poisoning.

Effects of aspirin and mefenamic acid on soman poisoning-induced neuropathology in mice.

The efficacy of aspirin and mefenamic acid to counteract soman-induced brain damage was investigated in mice. Neuronal damage was evaluated in the hippocampus and amygdala by performing omega3 receptor density measurements and hemalun-phloxin staining. The effect of both drugs on the proliferation of neural progenitors after soman exposure was also assessed. Mefenamic acid aggravated the soman-induced hippocampal neuropathology. On the other hand, aspirin recorded a weak neuroprotective effect in the amygdala. However, this drug also diminished the proliferation of neural precursor cells. The possible neurochemical mechanisms underlying such differences in the efficacy of the two drugs are also reviewed.

Early reduction of NeuN antigenicity induced by soman poisoning in mice can be used to predict delayed neuronal degeneration in the hippocampus.

The neuronal nuclei (NeuN) antigen is increasingly being used as a specific marker to identify neuronal cell loss under various pathological conditions. However, recent studies pointed out that a decrease in NeuN labeling could also be due to the reduction of protein expression level or loss of antigenicity and this was not necessarily related to neuronal cell disappearance. We also investigated the presence of damaged neurons, the loss of NeuN immunoreactivity and the level of NeuN protein in the brain hippocampus of mice subjected to soman poisoning (1.2 LD50 of soman). Damaged neurons were detected using hemalun-phloxin (H&P) and Fluoro-Jade B (FJB) staining on brain sections. NeuN immunohistochemistry was also performed on adjacent brain sections and NeuN protein level quantified by Western blot analysis. One and eight days after soman exposure, about 49% of hippocampal neurons were damaged, as assessed by H&P or FJB staining. NeuN immunohistochemistry indicated that all these damaged neurons were deprived of NeuN immunoreactivity. Using Western blot analysis, we proved that loss of NeuN immunoreactivity in degenerating neurons was due to reduced NeuN antigenicity rather than a fall in protein expression level. In this study, we discuss the potential use of NeuN immunohistochemistry as a good biomarker to predict delayed neuronal degeneration in the rodent hippocampus after various brain injuries.

Neuronal regeneration partially compensates the delayed neuronal cell death observed in the hippocampal CA1 field of soman-poisoned mice.

Soman poisoning induces long-term neuropathology characterized by the presence of damaged neurons up to 2 months after exposure in various central brain areas, especially the hippocampal CA1 layer. Rapid depletion of this layer could therefore be expected. Surprisingly, the CA1 layer remained consistently visible, suggesting delayed death of these damaged neurons, potentially accompanied by neuronal regeneration. To address this issue, mice were exposed to a convulsive dose of soman (110 microg/kg followed by 5.0mg/kg of atropine methyl nitrate (MNA) 1 min later) and brains were collected from day 1 to day 90 post-exposure. Damaged and residual healthy neurons were quantified on brain sections using hemalun-phloxin and fluorojade staining or neuronal nuclei antigen (NeuN) immunohistochemistry. On post-soman day 1, a moderate neuronal cell death was noticed in the hippocampal CA1 layer. In this area, an important and steady quantity of damaged neurons (about 48% of the whole pyramidal neurons) was detected from post-soman day 1 to day 30. Thus, throughout this period, damaged neurons seemed to survive, as confirmed by the unmodified depth of the hippocampal CA1 layer. The dramatic disappearance of the damaged neurons occurred only later during the experiment and was almost complete at day 90 after soman exposure. Interestingly, between day 30 and day 90 following poisoning, an increase in the number of residual healthy pyramidal neurons was observed. These different kinetic patterns related to the density of total, damaged and residual healthy neurons after soman poisoning demonstrate that neuronal regeneration is delayed in the hippocampal CA1 layer and is concomitant to the death of damaged neurons.

Efficacy of the ketamine-atropine combination in the delayed treatment of soman-induced status epilepticus.

Nerve agent poisoning is known to induce full-blown seizures, seizure-related brain damage (SRBD), and lethality. Effective and quick management of these seizures is critical. In conditions of delayed treatment, presently available measures are inadequate calling for optimization of therapeutic approaches. The effects of ketamine/atropine sulfate (KET/AS) combinations were thus assessed as potential valuable delayed therapy in soman-poisoned male guinea pigs. Animals received pyridostigmine (26 microg/kg, i.m.) 30 min before soman (62 microg/kg, i.m.) followed by therapy consisting of atropine methyl nitrate (4 mg/kg) 1 min later. KET was then administered i.m. at different times after the onset of seizures, starting at 30 min post-poisoning. KET was always injected with atropine sulfate, itself given at a dose that was unable to modify seizures (2 to 10 mg/kg). Different treatment schemes (dose and time of injection) were evaluated. Sub-anesthetic doses of KET (10 mg/kg) could prevent lethality and stop ongoing seizures only when administered 30 min after challenge. An extended delay before treatment (up to 2 h) called for an increase in KET dose (up to 60 mg/kg three times), thus reaching anesthetic levels but without the need of any ventilation support. KET proved effective in stopping seizures, highly reducing SRBD and allowing survival with a progressive loss of efficacy when treatment was delayed beyond 1 h post-challenge. Preliminary results suggest that association with the benzodiazepine midazolam (1 mg/kg) might be interesting when treatment is initiated 2 h after poisoning, i.e., when KET efficacy is dramatically reduced. All in all, these observations suggest that KET, in association with atropine sulfate and possibly other drugs, may be highly effective in the delayed treatment of severe soman intoxication.

Effect of cytokine treatment on the neurogenesis process in the brain of soman-poisoned mice.

We previously described that enhanced proliferation of neural progenitors occurred in the subgranular zone (SGZ) of the dentate gyrus and in the subventricular zone (SVZ) of the mouse brain following soman poisoning. Then, a discrete number of these cells seemed to migrate and engraft into the main damaged brain regions (hippocampus; septum and amygdala) and subsequently differentiate into neurons. In the present study, the effect of a cytokine treatment on the neurogenesis process was evaluated. For this purpose, subcutaneous injection of a cocktail of 40 microg/kg epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) was administered daily to soman-poisoned mice (110 microg/kg soman and 5.0 mg/kg methyl nitrate atropine), from post-soman days 1 to 8. To label replicating neural progenitors, 200 mg/kg bromodeoxyuridine (BrdU) was injected twice a day between post-soman days 6 and 8. Mice were sacrificed on post-soman day 9 or 34. On post-soman day 9, the cytokine treatment had no effect on the proliferation of neural progenitors in the SVZ and SGZ, as assessed by BrdU immunochemistry. However, this treatment seemed to promote the migration of neural precursor cells from the proliferative areas towards damaged brain regions. Indeed, in the CA1 hippocampal layer of soman-poisoned mice, on post-soman day 34, the cytokine treatment increased the number of healthy pyramidal neurons stained by hemalun-eosin dye. The cytokine treatment also augmented the number of BrdU-labeled cells in the CA1 hippocampal layer and amygdala. Interestingly, the administration of cytokines resulted in the differentiation of BrdU-positive cells into new neurons in the CA1 hippocampal layer, whereas astrocytic differentiation was preferentially observed in the amygdala.

Soman poisoning increases neural progenitor proliferation and induces long-term glial activation in mouse brain.

To date, only short-term glial reaction has been extensively studied following soman or other warfare neurotoxicant poisoning. In a context of cell therapy by neural progenitor engraftment to repair brain damage, the long-term effect of soman on glial reaction and neural progenitor division was analyzed in the present study. The effect of soman poisoning was estimated in mouse brains at various times ranging from 1 to 90 days post-poisoning. Using immunochemistry and dye staining techniques (hemalun-eosin staining), the number of degenerating neurons, the number of dividing neural progenitors, and microglial, astroglial or oligodendroglial cell activation were studied. Soman poisoning led to rapid and massive (post-soman day 1) death of mature neurons as assessed by hemalun-eosin staining. Following this acute poisoning phase, a weak toxicity effect on mature neurons was still observed for a period of 1 month after poisoning. A massive short-termed microgliosis peaked on day 3 post-poisoning. Delayed astrogliosis was observed from 3 to 90 days after soman poisoning, contributing to glial scar formation. On the other hand, oligodendroglial cells or their precursors were practically unaffected by soman poisoning. Interestingly, neural progenitors located in the subgranular zone of the dentate gyrus (SGZ) or in the subventricular zone (SVZ) of the brain survived soman poisoning. Furthermore, soman poisoning significantly increased neural progenitor proliferation in both SGZ and SVZ brain areas on post-soman day 3 or day 8, respectively. This increased proliferation rate was detected up to 1 month after poisoning.