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Neuronal regeneration partially compensates the delayed neuronal cell death observed in the hippocampal CA1 field of soman-poisoned mice
NeuroToxicology 27 (2006) 201–209
Neuronal regeneration partially compensates the delayed neuronal cell death observed in the hippocampal CA1 field of soman-poisoned mice Jean-Marc Collombet a,*, Pierre Carpentier a, Vale´rie Baille a, Elise Four a, Denis Bernabe´ b, Marie-France Burckhart b, Catherine Masqueliez a, Dominique Baubichon a, Guy Lallement a a
De´partement de Toxicologie, Centre de Recherches du Service de Sante´ des Arme´es (CRSSA), 24, avenue des Maquis du Gre´sivaudan, B.P. 87, 38702 LA TRONCHE cedex, France b Unite´ de Microscopie, CRSSA, 24, avenue des Maquis du Gre´sivaudan, B.P. 87, 38702 LA TRONCHE cedex, France Received 4 July 2005; accepted 10 October 2005 Available online 23 November 2005
Abstract 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 mg/kg followed by 5.0 mg/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. # 2005 Elsevier Inc. All rights reserved. Keywords: Soman; Hemalun-phloxin; Fluorojade; NeuN; Neuronal regeneration; Neurogenesis
1. Introduction Depending on the exposure dose, soman (pinacolyl methylphosphono-fluoridate) poisoning can lead to generalized convulsions and continuous epileptic seizures in animal models, lasting for several hours at least (Shih, 1993). This powerful warfare neurotoxicant acts as an irreversible cholinesterase inhibitor. Excitotoxicity resulting from cholinesterase inactivation is mainly due to the accumulation of
* Corresponding author. Tel.: +33 4 76 63 97 47; fax: +33 4 76 63 69 62. E-mail address: [email protected] (J.-M. Collombet). 0161-813X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2005.10.002
acetylcholine in the central nervous system (CNS) and subsequent glutamate release (Lallement et al., 1992). If a mixture of oxime (cholinesterase activator), atropine (muscarinic cholinergic antagonist) and diazepam (benzodiazepine anti-epileptic drug) is not rapidly administered to the poisoned animal (within the first 20 min following soman exposure), then irreversible brain lesions are observed (Lallement et al., 1998). Neuropathology has been extensively described in somanpoisoned rodents subjected or not to various supportive pretreatment (e.g. Lemercier et al., 1983; McLeod, 1985; Carpentier et al., 1990; Tryphonas and Clement, 1995; McDonough et al., 1998; Shih et al., 2003; Myhrer et al., 2005). In these studies, as assessed either by hemalun-phloxin
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or cresyl-violet staining performed on brain sections, significant neuronal cell death was generally detected from day 1 to day 3 following soman exposure, in all cortical areas, some subcortical limbic areas (amygdala, hippocampus, claustrum) and various thalamic nuclei. The severity and location of brain damage seemed to be correlated with the duration and intensity of seizure activity (Carpentier et al., 2000, 2001). To date, only a few studies have described the long-term evolution of neuropathology following soman poisoning (Lemercier et al., 1983; Kadar et al., 1992; McDonough et al., 1998; Collombet et al., 2005a). In all of them, numerous eosinophilic cells (damaged cells) were systematically detected in one or more of the above-mentioned cortical or subcortical areas and thalamic nuclei, up to 1 month after exposure. Focusing upon the hippocampal CA1 field, one of the most widely studied cerebral areas, after soman poisoning, Kadar et al. (1992) mentioned that ‘‘brain injury was characterized by the almost complete disappearance of the hippocampal CA1 cell layer’’ in rats, 3 months after 1.0 LD50 soman exposure without any protective treatment. In contradiction to this qualitative observation, the authors presented quantitative results proving that the number of CA1 cells fell significantly by 40% during the first post-poisoning week and the degenerative process continued at a much lower rate, finally recording a 50% reduction in the density of CA1 cells 3 months post-poisoning (Kadar et al., 1992). We recently described the qualitative evolution of cerebral lesions in the hippocampal CA1 layer of B6D2F1 mice intoxicated with 1.2 DL50 of soman and subjected to atropine methyl nitrate (MNA) supportive treatment (Collombet et al., 2005a). Major cell death was apparently observed on day 1 post-poisoning and a massive quantity of eosinophilic cells persisted in the CA1 area up to 1 month after poisoning. At later experimental times (3 months post-exposure), eosinophilic cells completely disappeared and there were apparently still large numbers of residual healthy cells in the CA1 area. In the same murin model of soman intoxication, we also showed that soman poisoning increased the proliferation rate of neural progenitors and these cells were able to migrate, engraft in the hippocampal CA1 field and subsequently differentiate into new neurons (Collombet et al., 2005b). Altogether, the relatively limited soman-induced brain damage observed 3 months after exposure by Kadar’s group and ourselves could result from neuronal regeneration in the CA1 area driven by a neurogenesis process. The purpose of the present study was to address this issue. For this purpose, the number of total, damaged and residual healthy cells was, respectively, quantified in the hippocampal CA1 layer of soman-poisoned mice, from 24 h to 3 months postexposure using hemalun-phloxin (H&P) and fluorojade B (FJB) staining. Neuron identification and quantification were assessed by neuronal nuclei antigen (NeuN) immunohistochemistry. 2. Materials and methods 2.1. Soman exposure and brain sample collection Nine-week-old adult male B6D2F1/j@rj mice (Janvier Laboratories, France) were housed in cages under standard conditions of temperature (24 8C) and humidity (50–60%),
with a 12:12-h light/dark cycle and free access to water and standard laboratory chow. All the experiments in this study were reviewed and approved by the Institutional Animal Care and Research Advisory Committee in accordance with French law and the main international guidelines. On day 0, soman (110 mg/kg in 200 ml of saline solution) – provided by the ‘‘Centre d’Etudes du Bouchet’’ (France) – was subcutaneously injected into the mice, followed 1 min later by an intraperitoneal injection of atropine methyl nitrate (MNA) at 5.0 mg/kg body weight (200 ml in saline). Only mice exhibiting well-characterized physical signs of convulsions, as previously described (Collombet et al., 2005a,c), were selected for the experiments. Indeed, according to numerous previous studies (Lallement et al., 1993; Carpentier et al., 2001), it is widely known that animals displaying convulsions for at least 2 h develop brain damage. Pentobarbital (80 mg/kg) anaesthetized mice were sacrificed on days 1, 3, 8, 15, 30, 60 and 90 post-poisoning (n = 7–12 animals per experimental time from two distinct experiments) by intracardiac perfusion of saline with heparin (5 UI/ml) followed by a fixative solution of formaldehyde (4%) and acetic acid (3%) in saline. Brains were collected, post-fixed in formaldehyde (4%) for 6 h and then processed for paraffin embedding. Coronal sections (6 mm thick) were serially cut with a microtome from 1.82 mm (hippocampus area) posterior to the bregma, as shown in the Franklin and Paxinos stereotaxic mouse brain atlas (Franklin and Paxinos, 1997). Similar conditions were applied to nine control mice (intraperitoneal injections of MNA), but the soman injection was replaced by a subcutaneous injection of saline. 2.2. H&P and FJB staining To detect eosinophilic and residual healthy cells, H&P (hemalun-phloxin) or FJB (fluorojade B) staining was performed on paraffin-embedded sections as previously described (Lillie and Fullmer, 1976; Schmued et al., 1997). 2.3. NeuN immunohistochemistry The neuronal nuclei antigen (NeuN) is a very specific protein only expressed in highly differentiated neurons. Therefore, NeuN immunochemistry was used to label and quantify CA1 pyramidal neurons. Briefly, sections were deparaffinized, rehydrated and incubated for 10 min at room temperature in TBS buffer (0.15 M NaCl; 0.1 M Tris–HCl, pH 7.5) containing 0.3% (v/v) H2O2, to block endogenous peroxidases. After saturation of unspecific sites in TBS-BSA (TBS containing 1% w/v BSA) for 1 h at room temperature, the sections were incubated overnight at +4 8C with the primary antibody directed against NeuN (1:400 mouse anti-NeuN in TBS-BSA; Chemicon). After three 15 min washings in TBS, the sections were treated with the secondary antibody (1:1000 biotinylated anti-mouse IgG in TBS-BSA; Vector) for 1 h at room temperature and, finally, labeling was revealed using Vectastain ABC Elite kit (Vector) and DAB (Sigma).
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2.4. Quantification and statistical analysis Quantitative evaluations of total, damaged and residual healthy cells were performed using a computerized image analysis system (SAMBA, Alcatel, France) linked to a CCD video camera-fitted microscope. For each mouse, cells were counted bilaterally in the apex of the CA1 field (about 15,000 mm2) from two non-adjacent sections and averaged. Then, for each experimental time, the mean value and its standard error (S.E.M.) were calculated for 7–12 animals (see above). To evaluate acute soman toxicity, neuronal density (total, degenerating or healthy cells) was compared between control and post-soman day 1 mice, using a Mann–Whitney U-test. Significance was set at p < 0.05. To appraise the evolution of cell density (total, degenerating or healthy cells) in poisoned mice over the time, statistical comparisons were achieved with Dunnett’s tests by comparing post-soman day 1 values (reference) with values from every other experimental time after intoxication. Significance was set at p < 0.05. All statistical comparisons were performed with Statistica 5.1 software (StatSoft, France). 3. Results 3.1. Analysis of H&P and fluorojade staining As assessed either by H&P or FJB staining, no eosinophilic cells were detected in the hippocampal CA1 layer of control mice (Figs. 1 and 2A). One day after soman exposure, a massive quantity of eosinophilic cells was revealed with H&P staining in the CA1 field (Fig. 1C). Eosinophilic cells appeared as reddish fuchsia shrunken cells with a highly condensed reddish violet nucleus. A few swollen non-eosinophilic cells were also spotted in the hippocampal CA1 layer. Important vacuolar edema was observed in the CA1 field and surrounding areas such as stratum oriens and stratum radiatum layers of the hippocampus (Fig. 1C). Using FJB staining, numerous fluorescent cells were detected confirming the presence of damaged cells at this early experimental time (Fig. 2B). Later, on post-soman day 30, the number of both H&P-stained eosinophilic cells and fluorescent FJB-stained cells was still very high (Figs. 1E and 2C). However, H&P-stained eosinophilic cells had a different morphology compared with their counterparts 1 day after soman exposure. Indeed, eosinophilic cells appeared to be ‘‘ghost’’ cells displaying homogeneous fuchsia stain where the limit between cytoplasm and nucleus was very difficult to appraise. On post-soman day 90, no more H&P-eosinophilic and fluorescent FJB-stained cells were detected (Figs. 1G and 2D). Furthermore, an obvious reduction in the depth of the hippocampal CA1 field seemed to be associated with the disappearance of these damaged cells. To assess these preliminary observations, total, eosinophilic and residual healthy cells revealed by H&P staining were quantified in the CA1 field of control and soman-poisoned mice with post-exposure times ranging from day 1 to day 90. Thus, 6590 300 total cells per mm2 were present in the hippocampal CA1 pyramidal layer of control mice. On post-
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soman day 1, total cell density significantly ( p < 0.05) decreased to 5390 210 total cells per mm2 (Fig. 3). This implies that about one-fifth of hippocampal CA1 cells died during the first 24 h following soman exposure. Then, the quantity of total CA1 cells rose to about 6200 cells per mm2 and this cell density was maintained from day 8 to day 30 postexposure. For later experimental times, the number of total CA1 cells fell dramatically to reach 3950 200 cells per mm2 ( p < 0.05 compared with post-soman day 1) on post-soman day 90 (Fig. 3). As shown in Fig. 3, from post-soman day 1 to day 30, the density of damaged eosinophilic cells was almost steady and was ranging from 2560 80 to 3020 230 cells per mm2 representing about 48% of the total CA1 cells. On post-soman day 60, a decrease in the number of damaged cells was initiated and led to almost complete depletion of these cells in the CA1 region on post-soman day 90 (170 110 damaged cells per mm2; p < 0.01 compared with post-soman day 1; Fig. 3). These results imply that eosinophilic cells survived in the hippocampal CA1 layer up to 2 months after soman exposure and their death took place later, between 60 and 90 days after poisoning. The death of eosinophilic cells could explain the reduction in CA1 field depth observed on post-soman day 90, as mentioned above (Fig. 1G). In the hippocampal CA1 layer, as early as 1 day postexposure, soman poisoning induced a massive 57% drop in the number of healthy cells (2830 220 healthy cells per mm2; p < 0.001 as compared to the control value mentioned above, i.e. 6590 300 total cells per mm2). This was concomitant with both the appearance of eosinophilic cells and early cell death resulting from the acute toxicity stage (Fig. 4). Between post-soman day 3 and day 15, the density of residual healthy cells increased slightly but continuously from 2990 156 to 3320 140 cells per mm2 (non-significant compared with post-soman day 1) as shown in Fig. 4. From post-soman day 30 to day 90, augmentation of the density of residual healthy cells in CA1 continued to become significant compared with the level measured 1 day after poisoning. Indeed, 30 days after exposure, 3540 170 residual healthy cells per mm2 were counted ( p < 0.05) while about 3750 residual healthy cells per mm2 were quantified on both day 60 and day 90 after soman exposure ( p < 0.01). 3.2. NeuN immunochemistry In the CA1 field of control mice, 6480 160 pyramidal neurons per mm2 were expressing NeuN (Fig. 1B). As mentioned above, a similar value was obtained for the number of healthy cells (6590 300 cells per mm2) detected by H&P staining in the control mice CA1. Soman poisoning induced a steep 58% fall in the number of NeuN-positive neurons in the CA1 as early as 1 day after intoxication (2720 160 NeuN-positive cells per mm2; p < 0.001 compared with the control mice; Figs. 1D and 4). The range of the fall in numbers of neurons expressing NeuN was equivalent to the one observed for residual healthy H&P-stained cells (57% as seen above). Then, the number of NeuN-labeled cells remained roughly unchanged up to post-soman day 15
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Fig. 1. Evolution of soman-induced neuropathology over time. Soman-poisoned mice were sacrificed on post-exposure days 1, 30 and 90. The collected brains were processed for hemalun-phloxin (H&P) staining (A, C, E and G) or anti-NeuN immunohistochemistry (B, D, F and H). Cells were viewed in the hippocampal CA1 layer under a light microscope. For H&P staining, damaged cells appeared in reddish fuchsia, while residual, healthy, unaffected cells were stained in white and violet. NeuN-positive neurons were stained in brown (DAB detection). Histological comparisons were made between control (A and B) and exposed mice on post-soman day 1 (C and D), day 30 (E and F) or day 90 (G and H). The same magnification was used for all the photographs (see scale bar on panel A).
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Fig. 2. Characterization of damaged neurons in the hippocampal CA1 layer of mice after soman exposure. Using fluorojade staining, damaged neurons were viewed in the hippocampal CA1 layer of brain sections from control (A) or poisoned mice on post-soman day 1 (B), day 30 (C) and day 90 (D). Only damaged neurons were fluorescent. Co-labeling of H&P staining with NeuN immunochemistry (DAB revelation) was performed in brain sections from intoxicated mice (post-soman day 8) to verify the identity of the damaged neurons (E). The same magnification was used for all the photographs (see scale bar on panel A).
(Fig. 4). For later experimental times, the density of CA1 NeuNlabeled neurons increased. This augmentation, which was very close to significance on post-soman day 30 and day 60, reached 3520 280 cells per mm2 on post-soman day 90, which is significantly higher than the density measured on post-soman day 1 ( p < 0.05; Figs. 1F and H and 4). Globally, the kinetic patterns of densities of both NeuNpositive neurons and residual healthy cells in the hippocampal CA1 field after poisoning were comparable. This suggests that H&P-stained healthy cells are almost exclusively NeuNexpressing neurons (even if it could not be excluded that a tiny number of other types of cerebral cells such as astrocytes, obviously deprived of NeuN expression, could be quantified as residual healthy H&P-stained cells).
To verify this hypothesis, a correlation study was performed and confirmed that there was significant linear correlation (correlation coefficient = 0.92; p < 0.001 using a t distribution test; Fig. 5) between the densities of both the NeuN-positive neurons and the residual healthy H&P-stained cells in the CA1 area of each control or soman-poisoned mouse. Therefore, it can be concluded that the great majority of healthy H&Pstained cells were pyramidal neurons expressing the NeuN antigen. This finding was confirmed by coupling H&P staining and NeuN immunochemistry on the same soman-poisoned mouse brain sections (mice sacrificed 8 days after soman exposure). As shown in Fig. 2E, only residual healthy H&Pstained cells expressed the NeuN protein, whereas eosinophilic cells did not.
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Fig. 3. Quantification of total and damaged cells in the mouse hippocampal CA1 layer after soman exposure. Total and damaged cells displayed by hemalun-phloxin staining in brain sections (see Fig. 1) from soman-poisoned mice on post-soman days 1, 3, 8, 15, 30, 60 or 90, were quantified using a computerized image analysis system (SAMBA, Alcatel, France) linked to a CCD video camera-equipped microscope. For each experimental group, the mean value for 7–12 animals and its standard error of the mean (S.E.M.) were calculated. Dunnett’s t-tests were performed to compare cell densities between post-soman day 1 (reference) and other experimental times after intoxication (*p < 0.05 and **p < 0.01).
Fig. 4. Quantification of NeuN-positive neurons and healthy hemalun-phloxinstained cells in the mouse hippocampal CA1 layer after soman exposure. NeuNpositive neurons and healthy hemalun-phloxin-stained cells were quantified in the hippocampus (CA1 layer) of soman-poisoned mice on days 1, 3, 8, 15, 30, 60 and 90 after exposure as mentioned in Section 2 (see also Fig. 3). For each experimental time, the values given represent the mean number of cells per mm2 and the standard error of the mean (S.E.M.) for 7–12 mice. Quantification between post-soman day 1 mice (reference) and poisoned mice at later experimental times was compared using Dunnett’s t-tests (*p < 0.05 and ** p < 0.01).
Fig. 5. Correlation between NeuN-positive neurons and healthy hemalunphloxin-stained cells in the hippocampus (CA1 layer) of control and somanexposed mice. Significance of the correlation coefficient was assessed using a t distribution test and was set at p < 0.05. ***p < 0.001.
3.3. Mathematical modeling of soman-induced cell regeneration A mathematical model for cell regeneration in the hippocampal CA1 layer after soman poisoning was built using the Kaleidagraph software (Synergy Software; USA). The kinetic patterns for the density of H&P-stained healthy cells could be included using a logarithmic regression curve as shown in Fig. 6.
Fig. 6. Kinetic pattern of cell regeneration in the mouse hippocampal CA1 layer following soman poisoning. The overtime evolution of the density of healthy hemalun-phloxin-stained neurons (see Fig. 4) was shown using a logarithmic regression curve. The curve equation is Y = 2769 + 512.72 log10 (X) where X is the number of days after soman poisoning and Y, the density of residual healthy neurons in the CA1 area. The regression coefficient of the curve is 0.99.
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4. Discussion In this study, we demonstrated that soman poisoning induced a three-step neuropathological process in the hippocampal CA1 layer of our B6D2F1 mouse model. In the first step, a wellknown early acute toxicity step (on post-soman day 1) was observed, leading to the rapid death of about one-fifth of the hippocampal CA1 neurons. At the same time, a massive quantity of damaged neurons (representing 48% of the total number of CA1 pyramidal neurons) appeared in the CA1 field. Then, these damaged neurons, deprived of NeuN immunoreactivity, survived for at least 2 months post-exposure, constituting the second step in the degenerative process that can be defined as a degenerative steady state. Finally, the third step consisted of delayed toxicity initiated on post-soman day 60 by slow death of the damaged eosinophilic neurons, leading to almost complete depletion of these neurons on post-soman day 90. While this degenerative process occurred, cell regeneration was detected in the CA1 region, characterized by the presence of new healthy cells that were certified as new neurons since they expressed the NeuN phenotype. This regeneration, which follows a logarithmic regression curve, led to a significant increase in density of these new neurons between post-soman day 30 and day 90 in the CA1 area. 4.1. Soman-induced long-term neuropathology To our knowledge, only a few studies have described longterm neuropathology following soman poisoning. Degenerating neurons were detected up to 39 days following exposure, in the hippocampal CA1 layer of rats subjected (McDonough et al., 1989, 1998) or not (Lemercier et al., 1983) to oxime supportive treatment. However, neuronal loss and the number of damaged neurons were not quantified in these studies. Kadar et al. (1992) performed this type of quantitative evaluation in somanpoisoned rats without supporting treatment, using H&P staining. The number of CA1 cells significantly decreased by about 40% over the first week after poisoning while the process continued at a slower rate, up to 3 months postexposure, leading to a final 50% cell loss. For the first time, in the present approach, a more complete kinetic study (seven experimental times tested) using more quantified parameters (total, damaged and residual healthy neurons with H&P staining or NeuN immunochemistry) is given. In our study, the detailed time-course of cell loss showed some differences with that observed by Kadar et al. (1992). Thus, compared with Kadar’s results, cell loss in our model appeared to be more discrete during the first week after poisoning (about 6% in our study versus 40% in Kadar’s study). The most massive reduction in the number of total cells and then thickness of the hippocampal CA1 layer was obtained, in the present study, between post-soman day 30 and day 90. This discrepancy between our results and those presented by Kadar et al. (1992) could be explained, as previously discussed (McDonough et al., 1998; Collombet et al., 2005a), either by the dose of injected soman or the type of supportive treatment administered or the species of animal models used. However, in both Kadar’s study
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and our present work, despite these discrepancies and differences in the neuronal degeneration kinetic pattern mentioned above, soman exposure led to a similar final reduction in depth of the CA1 field on post-soman day 90 and this was due to the death of about 45–50% of the pyramidal neurons. As mentioned above, different authors (Lemercier et al., 1983; McDonough et al., 1989, 1998; Collombet et al., 2005a) have observed damaged neurons in the CA1 region for several weeks following soman exposure. Due to the absence of quantitative evaluation, they could not reach any accurate conclusion concerning the potential variation in the numbers of damaged neurons over the period in question. In this study, we demonstrated that a steady elevated number of damaged neurons (about 48% of all the CA1 pyramidal neurons) was present in the CA1 region from post-soman day 1 to day 30. The death of these neurons was observed only at longer experimental times (i.e. between 30 and 90 days after exposure). This implies that the same damaged neurons survived during the first month after poisoning. Indeed, if these damaged neurons had a life expectancy of only a few days, complete erasure of the CA1 layer should be obtained rapidly, at least earlier than 2 months post-poisoning. Delayed neuronal death subsequent to the long-lasting presence of damaged neurons in the CA1 region of the rodent hippocampus has been already studied following kainate injections (Hopkins et al., 2000) or global and focal brain ischemia (Fukuda et al., 1993; Onodera et al., 1993; Wang et al., 2004). These two types of brain injuries involve glutamatergic over-stimulation leading to a neuronal death, which is similar to the neurodegenerative process induced by soman poisoning. Therefore, it is not surprising that such similarities exist in delayed neuronal death caused either by soman, kainate or ischemic insult. In this study, we also demonstrated that NeuN antigen is no longer detected in damaged neurons and this was shown very early following soman poisoning (i.e. 1 day post-exposure). Only residual healthy neurons expressed the NeuN antigen in our ¨ nal-Cevik et al. (2004) have already pointed out animal model. U this phenomenon in mice subjected to cerebral ischemia. The number of NeuN-labeled neurons in the brain fell 6 h after mild ischemic insult but H&P and Hoechst 33258 (detection of nucleated cells) staining revealed that these neurons still preserved their integrity and membrane intactness. Western blots showed that NeuN protein levels were not reduced, suggesting that decreased NeuN antigenicity accounted for loss ¨ nal-Cevik et al., 2004). A similar loss of of immunoreactivity (U immunoreactivity may have occurred in our soman-exposed animal model. Furthermore, NeuN immunochemistry appears to be a good biomarker for predicting long-term neuronal degeneration following soman exposure. Indeed, H&P or fluorojade-stained degenerating neurons destined to die several weeks after exposure are already NeuN negative as early as 1 day after exposure. Therefore, microscopic observation of the CA1 region on post-soman day 1 after NeuN immuno-labeling, should give a good indication of what the CA1 field thickness would look like several weeks later.
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4.2. Neuronal regeneration induced after soman poisoning The other original aspect of our approach was the demonstration of a significant increase in the number of healthy neurons in the CA1 region in soman-poisoned mice at late experimental time post-exposure, between post-soman day 30 and day 90. An active neurogenesis process could explain the observed cell regeneration. Neurogenesis results from the proliferation of neural progenitors located in the subgranular zone (SGZ) of the dentate gyrus and the subventricular zone (SVZ) of the brain (Lie et al., 2004). It has been shown, in various neuropathological conditions, that following enhanced proliferation, these progenitors migrate towards the site of lesions and subsequently differentiate mainly into neurons but also into astrocytes. The neuropathological conditions for which neurogenesis was described included focal (Nakatomi et al., 2002) or global (Schmidt and Reymann, 2002) ischemia and kainic acid- or pilocarpine-induced status epilepticus (Scharfman et al., 2000; Dong et al., 2003). In soman-poisoned mice, we previously demonstrated that neurotoxicant exposure expanded SVZ and SGZ neural progenitor populations in the mouse brain from post-soman day 8 to day 30 (Collombet et al., 2005a). These newly generated progenitors then migrated from proliferative regions to sites of severe brain damage such as the hippocampal CA1 layer and the amygdala (Collombet et al., 2005a,b). Finally, when engrafted in lesioned cerebral areas, neural precursors preferentially differentiated into neurons in the CA1 region or astrocytes in the amygdala (Collombet et al., 2005b). These new neurons and astrocytes were detected as early as 34 days post-exposure. Therefore, the results presented in this study are consistent with all our previously published observations. Indeed, the CA1 neuronal regeneration revealed between days 60 and days 90 after soman exposure and attested by the presence of new healthy neurons, is compatible with enhanced proliferation of neural progenitors (detected up to post-soman day 30) followed by their subsequent migration and differentiation into new neurons at later experimental times. As additional evidence, we applied the present mathematical equation of the logarithmic regression curve modeling the CA1 neuronal regeneration (see Fig. 6) to our previously published independent histological data (Collombet et al., 2005b). This equation allows us to predict the number of mature healthy neurons in the CA1 region following soman exposure in our animal model at a given time. When applied to our previously published data arguing in favor of a neurogenesis process (Collombet et al., 2005b), the accuracy of the present mathematical prediction was higher than 97%, reinforcing the validity of the neurogenesis hypothesis. However, an alternative hypothesis, whereby a reversion status of degenerating neurons reverting to healthy ones may also be partially involved in CA1 neuronal regeneration, could not be totally excluded. In conclusion, for the first time, this study demonstrates delayed neuronal death in the cerebral hippocampal CA1 layer of our soman-intoxicated mouse model, accompanied by neuronal regeneration. Arguments are strongly in favor of a
neurogenesis process to explain this regeneration but the hypothesis of a reversion event with degenerating neurons reverting to healthy neurons cannot be completely ruled out.
Acknowledgments The authors would like to thank Dr. Agne`s JOB (CRSSA) for her helpful advice regarding the statistical treatment of our data. This work is supported by a grant from the DGA-DSPSTTC-SH (De´le´gation Ge´ne´rale pour l’Armement; grant No. 02 CO 006) of the French Ministry of Defense.
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Scharfman HE, Goodman JH, Sollas AL. Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis. J Neurosci 2000;20(15):6144–58. Shih TM, Duniho SM, McDonough JH. Control of nerve agent-induced seizures is critical for neuroprotection and survival. Toxicol appl Pharmacol 2003;188:69–80. Shih TM. Comparison of several oximes on reactivation of soman-inhibited blood, brain and tissue cholinesterase activity in rats. Arch Toxicol 1993;67:637–46. Schmidt W, Reymann KG. Proliferating cells differentiate into neurons in the hippocampal CA1 region of gerbils after global cerebral ischemia. Neurosci Lett 2002;334:153–6. Schmued LC, Albertson C, Slikker W. Fluoro-jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res 1997;751:37–46. Tryphonas L, Clement JG. Histomorphogenesis of soman-induced encephalocardiomyopathy in Sprague-Dawley rats. Toxicol Pathol 1995;23:393–409. ¨ zdemir Y, Gurer G, Dalkara T. Loss of NeuN ¨ nal-Cevik I, Kilinc¸ M, Gu¨rsoy-O U immunoreactivity after cerebral ischemia does not indicate neuronal cell loss: a cautionary note. Brain Res 2004;1015:169–74. Wang W, Redecker C, Bidmon HJ, Witte OW. Delayed neuronal death and damage of GDNF family receptors in CA1 following focal cerebral ischemia. Brain Res 2004;1023:92–101.
Neuronal regeneration partially compensates the delayed neuronal cell death observed in the hippocampal CA1 field of soman-poisoned mice Jean-Marc Collombet a,*, Pierre Carpentier a, Vale´rie Baille a, Elise Four a, Denis Bernabe´ b, Marie-France Burckhart b, Catherine Masqueliez a, Dominique Baubichon a, Guy Lallement a a
De´partement de Toxicologie, Centre de Recherches du Service de Sante´ des Arme´es (CRSSA), 24, avenue des Maquis du Gre´sivaudan, B.P. 87, 38702 LA TRONCHE cedex, France b Unite´ de Microscopie, CRSSA, 24, avenue des Maquis du Gre´sivaudan, B.P. 87, 38702 LA TRONCHE cedex, France Received 4 July 2005; accepted 10 October 2005 Available online 23 November 2005
Abstract 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 mg/kg followed by 5.0 mg/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. # 2005 Elsevier Inc. All rights reserved. Keywords: Soman; Hemalun-phloxin; Fluorojade; NeuN; Neuronal regeneration; Neurogenesis
1. Introduction Depending on the exposure dose, soman (pinacolyl methylphosphono-fluoridate) poisoning can lead to generalized convulsions and continuous epileptic seizures in animal models, lasting for several hours at least (Shih, 1993). This powerful warfare neurotoxicant acts as an irreversible cholinesterase inhibitor. Excitotoxicity resulting from cholinesterase inactivation is mainly due to the accumulation of
* Corresponding author. Tel.: +33 4 76 63 97 47; fax: +33 4 76 63 69 62. E-mail address: [email protected] (J.-M. Collombet). 0161-813X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2005.10.002
acetylcholine in the central nervous system (CNS) and subsequent glutamate release (Lallement et al., 1992). If a mixture of oxime (cholinesterase activator), atropine (muscarinic cholinergic antagonist) and diazepam (benzodiazepine anti-epileptic drug) is not rapidly administered to the poisoned animal (within the first 20 min following soman exposure), then irreversible brain lesions are observed (Lallement et al., 1998). Neuropathology has been extensively described in somanpoisoned rodents subjected or not to various supportive pretreatment (e.g. Lemercier et al., 1983; McLeod, 1985; Carpentier et al., 1990; Tryphonas and Clement, 1995; McDonough et al., 1998; Shih et al., 2003; Myhrer et al., 2005). In these studies, as assessed either by hemalun-phloxin
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or cresyl-violet staining performed on brain sections, significant neuronal cell death was generally detected from day 1 to day 3 following soman exposure, in all cortical areas, some subcortical limbic areas (amygdala, hippocampus, claustrum) and various thalamic nuclei. The severity and location of brain damage seemed to be correlated with the duration and intensity of seizure activity (Carpentier et al., 2000, 2001). To date, only a few studies have described the long-term evolution of neuropathology following soman poisoning (Lemercier et al., 1983; Kadar et al., 1992; McDonough et al., 1998; Collombet et al., 2005a). In all of them, numerous eosinophilic cells (damaged cells) were systematically detected in one or more of the above-mentioned cortical or subcortical areas and thalamic nuclei, up to 1 month after exposure. Focusing upon the hippocampal CA1 field, one of the most widely studied cerebral areas, after soman poisoning, Kadar et al. (1992) mentioned that ‘‘brain injury was characterized by the almost complete disappearance of the hippocampal CA1 cell layer’’ in rats, 3 months after 1.0 LD50 soman exposure without any protective treatment. In contradiction to this qualitative observation, the authors presented quantitative results proving that the number of CA1 cells fell significantly by 40% during the first post-poisoning week and the degenerative process continued at a much lower rate, finally recording a 50% reduction in the density of CA1 cells 3 months post-poisoning (Kadar et al., 1992). We recently described the qualitative evolution of cerebral lesions in the hippocampal CA1 layer of B6D2F1 mice intoxicated with 1.2 DL50 of soman and subjected to atropine methyl nitrate (MNA) supportive treatment (Collombet et al., 2005a). Major cell death was apparently observed on day 1 post-poisoning and a massive quantity of eosinophilic cells persisted in the CA1 area up to 1 month after poisoning. At later experimental times (3 months post-exposure), eosinophilic cells completely disappeared and there were apparently still large numbers of residual healthy cells in the CA1 area. In the same murin model of soman intoxication, we also showed that soman poisoning increased the proliferation rate of neural progenitors and these cells were able to migrate, engraft in the hippocampal CA1 field and subsequently differentiate into new neurons (Collombet et al., 2005b). Altogether, the relatively limited soman-induced brain damage observed 3 months after exposure by Kadar’s group and ourselves could result from neuronal regeneration in the CA1 area driven by a neurogenesis process. The purpose of the present study was to address this issue. For this purpose, the number of total, damaged and residual healthy cells was, respectively, quantified in the hippocampal CA1 layer of soman-poisoned mice, from 24 h to 3 months postexposure using hemalun-phloxin (H&P) and fluorojade B (FJB) staining. Neuron identification and quantification were assessed by neuronal nuclei antigen (NeuN) immunohistochemistry. 2. Materials and methods 2.1. Soman exposure and brain sample collection Nine-week-old adult male B6D2F1/j@rj mice (Janvier Laboratories, France) were housed in cages under standard conditions of temperature (24 8C) and humidity (50–60%),
with a 12:12-h light/dark cycle and free access to water and standard laboratory chow. All the experiments in this study were reviewed and approved by the Institutional Animal Care and Research Advisory Committee in accordance with French law and the main international guidelines. On day 0, soman (110 mg/kg in 200 ml of saline solution) – provided by the ‘‘Centre d’Etudes du Bouchet’’ (France) – was subcutaneously injected into the mice, followed 1 min later by an intraperitoneal injection of atropine methyl nitrate (MNA) at 5.0 mg/kg body weight (200 ml in saline). Only mice exhibiting well-characterized physical signs of convulsions, as previously described (Collombet et al., 2005a,c), were selected for the experiments. Indeed, according to numerous previous studies (Lallement et al., 1993; Carpentier et al., 2001), it is widely known that animals displaying convulsions for at least 2 h develop brain damage. Pentobarbital (80 mg/kg) anaesthetized mice were sacrificed on days 1, 3, 8, 15, 30, 60 and 90 post-poisoning (n = 7–12 animals per experimental time from two distinct experiments) by intracardiac perfusion of saline with heparin (5 UI/ml) followed by a fixative solution of formaldehyde (4%) and acetic acid (3%) in saline. Brains were collected, post-fixed in formaldehyde (4%) for 6 h and then processed for paraffin embedding. Coronal sections (6 mm thick) were serially cut with a microtome from 1.82 mm (hippocampus area) posterior to the bregma, as shown in the Franklin and Paxinos stereotaxic mouse brain atlas (Franklin and Paxinos, 1997). Similar conditions were applied to nine control mice (intraperitoneal injections of MNA), but the soman injection was replaced by a subcutaneous injection of saline. 2.2. H&P and FJB staining To detect eosinophilic and residual healthy cells, H&P (hemalun-phloxin) or FJB (fluorojade B) staining was performed on paraffin-embedded sections as previously described (Lillie and Fullmer, 1976; Schmued et al., 1997). 2.3. NeuN immunohistochemistry The neuronal nuclei antigen (NeuN) is a very specific protein only expressed in highly differentiated neurons. Therefore, NeuN immunochemistry was used to label and quantify CA1 pyramidal neurons. Briefly, sections were deparaffinized, rehydrated and incubated for 10 min at room temperature in TBS buffer (0.15 M NaCl; 0.1 M Tris–HCl, pH 7.5) containing 0.3% (v/v) H2O2, to block endogenous peroxidases. After saturation of unspecific sites in TBS-BSA (TBS containing 1% w/v BSA) for 1 h at room temperature, the sections were incubated overnight at +4 8C with the primary antibody directed against NeuN (1:400 mouse anti-NeuN in TBS-BSA; Chemicon). After three 15 min washings in TBS, the sections were treated with the secondary antibody (1:1000 biotinylated anti-mouse IgG in TBS-BSA; Vector) for 1 h at room temperature and, finally, labeling was revealed using Vectastain ABC Elite kit (Vector) and DAB (Sigma).
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2.4. Quantification and statistical analysis Quantitative evaluations of total, damaged and residual healthy cells were performed using a computerized image analysis system (SAMBA, Alcatel, France) linked to a CCD video camera-fitted microscope. For each mouse, cells were counted bilaterally in the apex of the CA1 field (about 15,000 mm2) from two non-adjacent sections and averaged. Then, for each experimental time, the mean value and its standard error (S.E.M.) were calculated for 7–12 animals (see above). To evaluate acute soman toxicity, neuronal density (total, degenerating or healthy cells) was compared between control and post-soman day 1 mice, using a Mann–Whitney U-test. Significance was set at p < 0.05. To appraise the evolution of cell density (total, degenerating or healthy cells) in poisoned mice over the time, statistical comparisons were achieved with Dunnett’s tests by comparing post-soman day 1 values (reference) with values from every other experimental time after intoxication. Significance was set at p < 0.05. All statistical comparisons were performed with Statistica 5.1 software (StatSoft, France). 3. Results 3.1. Analysis of H&P and fluorojade staining As assessed either by H&P or FJB staining, no eosinophilic cells were detected in the hippocampal CA1 layer of control mice (Figs. 1 and 2A). One day after soman exposure, a massive quantity of eosinophilic cells was revealed with H&P staining in the CA1 field (Fig. 1C). Eosinophilic cells appeared as reddish fuchsia shrunken cells with a highly condensed reddish violet nucleus. A few swollen non-eosinophilic cells were also spotted in the hippocampal CA1 layer. Important vacuolar edema was observed in the CA1 field and surrounding areas such as stratum oriens and stratum radiatum layers of the hippocampus (Fig. 1C). Using FJB staining, numerous fluorescent cells were detected confirming the presence of damaged cells at this early experimental time (Fig. 2B). Later, on post-soman day 30, the number of both H&P-stained eosinophilic cells and fluorescent FJB-stained cells was still very high (Figs. 1E and 2C). However, H&P-stained eosinophilic cells had a different morphology compared with their counterparts 1 day after soman exposure. Indeed, eosinophilic cells appeared to be ‘‘ghost’’ cells displaying homogeneous fuchsia stain where the limit between cytoplasm and nucleus was very difficult to appraise. On post-soman day 90, no more H&P-eosinophilic and fluorescent FJB-stained cells were detected (Figs. 1G and 2D). Furthermore, an obvious reduction in the depth of the hippocampal CA1 field seemed to be associated with the disappearance of these damaged cells. To assess these preliminary observations, total, eosinophilic and residual healthy cells revealed by H&P staining were quantified in the CA1 field of control and soman-poisoned mice with post-exposure times ranging from day 1 to day 90. Thus, 6590 300 total cells per mm2 were present in the hippocampal CA1 pyramidal layer of control mice. On post-
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soman day 1, total cell density significantly ( p < 0.05) decreased to 5390 210 total cells per mm2 (Fig. 3). This implies that about one-fifth of hippocampal CA1 cells died during the first 24 h following soman exposure. Then, the quantity of total CA1 cells rose to about 6200 cells per mm2 and this cell density was maintained from day 8 to day 30 postexposure. For later experimental times, the number of total CA1 cells fell dramatically to reach 3950 200 cells per mm2 ( p < 0.05 compared with post-soman day 1) on post-soman day 90 (Fig. 3). As shown in Fig. 3, from post-soman day 1 to day 30, the density of damaged eosinophilic cells was almost steady and was ranging from 2560 80 to 3020 230 cells per mm2 representing about 48% of the total CA1 cells. On post-soman day 60, a decrease in the number of damaged cells was initiated and led to almost complete depletion of these cells in the CA1 region on post-soman day 90 (170 110 damaged cells per mm2; p < 0.01 compared with post-soman day 1; Fig. 3). These results imply that eosinophilic cells survived in the hippocampal CA1 layer up to 2 months after soman exposure and their death took place later, between 60 and 90 days after poisoning. The death of eosinophilic cells could explain the reduction in CA1 field depth observed on post-soman day 90, as mentioned above (Fig. 1G). In the hippocampal CA1 layer, as early as 1 day postexposure, soman poisoning induced a massive 57% drop in the number of healthy cells (2830 220 healthy cells per mm2; p < 0.001 as compared to the control value mentioned above, i.e. 6590 300 total cells per mm2). This was concomitant with both the appearance of eosinophilic cells and early cell death resulting from the acute toxicity stage (Fig. 4). Between post-soman day 3 and day 15, the density of residual healthy cells increased slightly but continuously from 2990 156 to 3320 140 cells per mm2 (non-significant compared with post-soman day 1) as shown in Fig. 4. From post-soman day 30 to day 90, augmentation of the density of residual healthy cells in CA1 continued to become significant compared with the level measured 1 day after poisoning. Indeed, 30 days after exposure, 3540 170 residual healthy cells per mm2 were counted ( p < 0.05) while about 3750 residual healthy cells per mm2 were quantified on both day 60 and day 90 after soman exposure ( p < 0.01). 3.2. NeuN immunochemistry In the CA1 field of control mice, 6480 160 pyramidal neurons per mm2 were expressing NeuN (Fig. 1B). As mentioned above, a similar value was obtained for the number of healthy cells (6590 300 cells per mm2) detected by H&P staining in the control mice CA1. Soman poisoning induced a steep 58% fall in the number of NeuN-positive neurons in the CA1 as early as 1 day after intoxication (2720 160 NeuN-positive cells per mm2; p < 0.001 compared with the control mice; Figs. 1D and 4). The range of the fall in numbers of neurons expressing NeuN was equivalent to the one observed for residual healthy H&P-stained cells (57% as seen above). Then, the number of NeuN-labeled cells remained roughly unchanged up to post-soman day 15
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Fig. 1. Evolution of soman-induced neuropathology over time. Soman-poisoned mice were sacrificed on post-exposure days 1, 30 and 90. The collected brains were processed for hemalun-phloxin (H&P) staining (A, C, E and G) or anti-NeuN immunohistochemistry (B, D, F and H). Cells were viewed in the hippocampal CA1 layer under a light microscope. For H&P staining, damaged cells appeared in reddish fuchsia, while residual, healthy, unaffected cells were stained in white and violet. NeuN-positive neurons were stained in brown (DAB detection). Histological comparisons were made between control (A and B) and exposed mice on post-soman day 1 (C and D), day 30 (E and F) or day 90 (G and H). The same magnification was used for all the photographs (see scale bar on panel A).
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Fig. 2. Characterization of damaged neurons in the hippocampal CA1 layer of mice after soman exposure. Using fluorojade staining, damaged neurons were viewed in the hippocampal CA1 layer of brain sections from control (A) or poisoned mice on post-soman day 1 (B), day 30 (C) and day 90 (D). Only damaged neurons were fluorescent. Co-labeling of H&P staining with NeuN immunochemistry (DAB revelation) was performed in brain sections from intoxicated mice (post-soman day 8) to verify the identity of the damaged neurons (E). The same magnification was used for all the photographs (see scale bar on panel A).
(Fig. 4). For later experimental times, the density of CA1 NeuNlabeled neurons increased. This augmentation, which was very close to significance on post-soman day 30 and day 60, reached 3520 280 cells per mm2 on post-soman day 90, which is significantly higher than the density measured on post-soman day 1 ( p < 0.05; Figs. 1F and H and 4). Globally, the kinetic patterns of densities of both NeuNpositive neurons and residual healthy cells in the hippocampal CA1 field after poisoning were comparable. This suggests that H&P-stained healthy cells are almost exclusively NeuNexpressing neurons (even if it could not be excluded that a tiny number of other types of cerebral cells such as astrocytes, obviously deprived of NeuN expression, could be quantified as residual healthy H&P-stained cells).
To verify this hypothesis, a correlation study was performed and confirmed that there was significant linear correlation (correlation coefficient = 0.92; p < 0.001 using a t distribution test; Fig. 5) between the densities of both the NeuN-positive neurons and the residual healthy H&P-stained cells in the CA1 area of each control or soman-poisoned mouse. Therefore, it can be concluded that the great majority of healthy H&Pstained cells were pyramidal neurons expressing the NeuN antigen. This finding was confirmed by coupling H&P staining and NeuN immunochemistry on the same soman-poisoned mouse brain sections (mice sacrificed 8 days after soman exposure). As shown in Fig. 2E, only residual healthy H&Pstained cells expressed the NeuN protein, whereas eosinophilic cells did not.
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Fig. 3. Quantification of total and damaged cells in the mouse hippocampal CA1 layer after soman exposure. Total and damaged cells displayed by hemalun-phloxin staining in brain sections (see Fig. 1) from soman-poisoned mice on post-soman days 1, 3, 8, 15, 30, 60 or 90, were quantified using a computerized image analysis system (SAMBA, Alcatel, France) linked to a CCD video camera-equipped microscope. For each experimental group, the mean value for 7–12 animals and its standard error of the mean (S.E.M.) were calculated. Dunnett’s t-tests were performed to compare cell densities between post-soman day 1 (reference) and other experimental times after intoxication (*p < 0.05 and **p < 0.01).
Fig. 4. Quantification of NeuN-positive neurons and healthy hemalun-phloxinstained cells in the mouse hippocampal CA1 layer after soman exposure. NeuNpositive neurons and healthy hemalun-phloxin-stained cells were quantified in the hippocampus (CA1 layer) of soman-poisoned mice on days 1, 3, 8, 15, 30, 60 and 90 after exposure as mentioned in Section 2 (see also Fig. 3). For each experimental time, the values given represent the mean number of cells per mm2 and the standard error of the mean (S.E.M.) for 7–12 mice. Quantification between post-soman day 1 mice (reference) and poisoned mice at later experimental times was compared using Dunnett’s t-tests (*p < 0.05 and ** p < 0.01).
Fig. 5. Correlation between NeuN-positive neurons and healthy hemalunphloxin-stained cells in the hippocampus (CA1 layer) of control and somanexposed mice. Significance of the correlation coefficient was assessed using a t distribution test and was set at p < 0.05. ***p < 0.001.
3.3. Mathematical modeling of soman-induced cell regeneration A mathematical model for cell regeneration in the hippocampal CA1 layer after soman poisoning was built using the Kaleidagraph software (Synergy Software; USA). The kinetic patterns for the density of H&P-stained healthy cells could be included using a logarithmic regression curve as shown in Fig. 6.
Fig. 6. Kinetic pattern of cell regeneration in the mouse hippocampal CA1 layer following soman poisoning. The overtime evolution of the density of healthy hemalun-phloxin-stained neurons (see Fig. 4) was shown using a logarithmic regression curve. The curve equation is Y = 2769 + 512.72 log10 (X) where X is the number of days after soman poisoning and Y, the density of residual healthy neurons in the CA1 area. The regression coefficient of the curve is 0.99.
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4. Discussion In this study, we demonstrated that soman poisoning induced a three-step neuropathological process in the hippocampal CA1 layer of our B6D2F1 mouse model. In the first step, a wellknown early acute toxicity step (on post-soman day 1) was observed, leading to the rapid death of about one-fifth of the hippocampal CA1 neurons. At the same time, a massive quantity of damaged neurons (representing 48% of the total number of CA1 pyramidal neurons) appeared in the CA1 field. Then, these damaged neurons, deprived of NeuN immunoreactivity, survived for at least 2 months post-exposure, constituting the second step in the degenerative process that can be defined as a degenerative steady state. Finally, the third step consisted of delayed toxicity initiated on post-soman day 60 by slow death of the damaged eosinophilic neurons, leading to almost complete depletion of these neurons on post-soman day 90. While this degenerative process occurred, cell regeneration was detected in the CA1 region, characterized by the presence of new healthy cells that were certified as new neurons since they expressed the NeuN phenotype. This regeneration, which follows a logarithmic regression curve, led to a significant increase in density of these new neurons between post-soman day 30 and day 90 in the CA1 area. 4.1. Soman-induced long-term neuropathology To our knowledge, only a few studies have described longterm neuropathology following soman poisoning. Degenerating neurons were detected up to 39 days following exposure, in the hippocampal CA1 layer of rats subjected (McDonough et al., 1989, 1998) or not (Lemercier et al., 1983) to oxime supportive treatment. However, neuronal loss and the number of damaged neurons were not quantified in these studies. Kadar et al. (1992) performed this type of quantitative evaluation in somanpoisoned rats without supporting treatment, using H&P staining. The number of CA1 cells significantly decreased by about 40% over the first week after poisoning while the process continued at a slower rate, up to 3 months postexposure, leading to a final 50% cell loss. For the first time, in the present approach, a more complete kinetic study (seven experimental times tested) using more quantified parameters (total, damaged and residual healthy neurons with H&P staining or NeuN immunochemistry) is given. In our study, the detailed time-course of cell loss showed some differences with that observed by Kadar et al. (1992). Thus, compared with Kadar’s results, cell loss in our model appeared to be more discrete during the first week after poisoning (about 6% in our study versus 40% in Kadar’s study). The most massive reduction in the number of total cells and then thickness of the hippocampal CA1 layer was obtained, in the present study, between post-soman day 30 and day 90. This discrepancy between our results and those presented by Kadar et al. (1992) could be explained, as previously discussed (McDonough et al., 1998; Collombet et al., 2005a), either by the dose of injected soman or the type of supportive treatment administered or the species of animal models used. However, in both Kadar’s study
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and our present work, despite these discrepancies and differences in the neuronal degeneration kinetic pattern mentioned above, soman exposure led to a similar final reduction in depth of the CA1 field on post-soman day 90 and this was due to the death of about 45–50% of the pyramidal neurons. As mentioned above, different authors (Lemercier et al., 1983; McDonough et al., 1989, 1998; Collombet et al., 2005a) have observed damaged neurons in the CA1 region for several weeks following soman exposure. Due to the absence of quantitative evaluation, they could not reach any accurate conclusion concerning the potential variation in the numbers of damaged neurons over the period in question. In this study, we demonstrated that a steady elevated number of damaged neurons (about 48% of all the CA1 pyramidal neurons) was present in the CA1 region from post-soman day 1 to day 30. The death of these neurons was observed only at longer experimental times (i.e. between 30 and 90 days after exposure). This implies that the same damaged neurons survived during the first month after poisoning. Indeed, if these damaged neurons had a life expectancy of only a few days, complete erasure of the CA1 layer should be obtained rapidly, at least earlier than 2 months post-poisoning. Delayed neuronal death subsequent to the long-lasting presence of damaged neurons in the CA1 region of the rodent hippocampus has been already studied following kainate injections (Hopkins et al., 2000) or global and focal brain ischemia (Fukuda et al., 1993; Onodera et al., 1993; Wang et al., 2004). These two types of brain injuries involve glutamatergic over-stimulation leading to a neuronal death, which is similar to the neurodegenerative process induced by soman poisoning. Therefore, it is not surprising that such similarities exist in delayed neuronal death caused either by soman, kainate or ischemic insult. In this study, we also demonstrated that NeuN antigen is no longer detected in damaged neurons and this was shown very early following soman poisoning (i.e. 1 day post-exposure). Only residual healthy neurons expressed the NeuN antigen in our ¨ nal-Cevik et al. (2004) have already pointed out animal model. U this phenomenon in mice subjected to cerebral ischemia. The number of NeuN-labeled neurons in the brain fell 6 h after mild ischemic insult but H&P and Hoechst 33258 (detection of nucleated cells) staining revealed that these neurons still preserved their integrity and membrane intactness. Western blots showed that NeuN protein levels were not reduced, suggesting that decreased NeuN antigenicity accounted for loss ¨ nal-Cevik et al., 2004). A similar loss of of immunoreactivity (U immunoreactivity may have occurred in our soman-exposed animal model. Furthermore, NeuN immunochemistry appears to be a good biomarker for predicting long-term neuronal degeneration following soman exposure. Indeed, H&P or fluorojade-stained degenerating neurons destined to die several weeks after exposure are already NeuN negative as early as 1 day after exposure. Therefore, microscopic observation of the CA1 region on post-soman day 1 after NeuN immuno-labeling, should give a good indication of what the CA1 field thickness would look like several weeks later.
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4.2. Neuronal regeneration induced after soman poisoning The other original aspect of our approach was the demonstration of a significant increase in the number of healthy neurons in the CA1 region in soman-poisoned mice at late experimental time post-exposure, between post-soman day 30 and day 90. An active neurogenesis process could explain the observed cell regeneration. Neurogenesis results from the proliferation of neural progenitors located in the subgranular zone (SGZ) of the dentate gyrus and the subventricular zone (SVZ) of the brain (Lie et al., 2004). It has been shown, in various neuropathological conditions, that following enhanced proliferation, these progenitors migrate towards the site of lesions and subsequently differentiate mainly into neurons but also into astrocytes. The neuropathological conditions for which neurogenesis was described included focal (Nakatomi et al., 2002) or global (Schmidt and Reymann, 2002) ischemia and kainic acid- or pilocarpine-induced status epilepticus (Scharfman et al., 2000; Dong et al., 2003). In soman-poisoned mice, we previously demonstrated that neurotoxicant exposure expanded SVZ and SGZ neural progenitor populations in the mouse brain from post-soman day 8 to day 30 (Collombet et al., 2005a). These newly generated progenitors then migrated from proliferative regions to sites of severe brain damage such as the hippocampal CA1 layer and the amygdala (Collombet et al., 2005a,b). Finally, when engrafted in lesioned cerebral areas, neural precursors preferentially differentiated into neurons in the CA1 region or astrocytes in the amygdala (Collombet et al., 2005b). These new neurons and astrocytes were detected as early as 34 days post-exposure. Therefore, the results presented in this study are consistent with all our previously published observations. Indeed, the CA1 neuronal regeneration revealed between days 60 and days 90 after soman exposure and attested by the presence of new healthy neurons, is compatible with enhanced proliferation of neural progenitors (detected up to post-soman day 30) followed by their subsequent migration and differentiation into new neurons at later experimental times. As additional evidence, we applied the present mathematical equation of the logarithmic regression curve modeling the CA1 neuronal regeneration (see Fig. 6) to our previously published independent histological data (Collombet et al., 2005b). This equation allows us to predict the number of mature healthy neurons in the CA1 region following soman exposure in our animal model at a given time. When applied to our previously published data arguing in favor of a neurogenesis process (Collombet et al., 2005b), the accuracy of the present mathematical prediction was higher than 97%, reinforcing the validity of the neurogenesis hypothesis. However, an alternative hypothesis, whereby a reversion status of degenerating neurons reverting to healthy ones may also be partially involved in CA1 neuronal regeneration, could not be totally excluded. In conclusion, for the first time, this study demonstrates delayed neuronal death in the cerebral hippocampal CA1 layer of our soman-intoxicated mouse model, accompanied by neuronal regeneration. Arguments are strongly in favor of a
neurogenesis process to explain this regeneration but the hypothesis of a reversion event with degenerating neurons reverting to healthy neurons cannot be completely ruled out.
Acknowledgments The authors would like to thank Dr. Agne`s JOB (CRSSA) for her helpful advice regarding the statistical treatment of our data. This work is supported by a grant from the DGA-DSPSTTC-SH (De´le´gation Ge´ne´rale pour l’Armement; grant No. 02 CO 006) of the French Ministry of Defense.
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