Galantamine counteracts development of learning impairment in guinea pigs exposed to the organophosphorus poison soman: clinical significance

Abstract

Galantamine, a drug used to treat Alzheimer's disease, protects guinea pigs against the acute toxicity and lethality of organophosphorus (OP) compounds, including soman. Here, we tested the hypothesis that a single exposure of guinea pigs to 1xLD50 soman triggers cognitive impairments that can be counteracted by galantamine. Thus, animals were injected intramuscularly with saline (0.5 ml/kg) or galantamine (8 mg/kg) and 30 min later injected subcutaneously with soman (26.3 μg/kg) or saline. Cognitive performance was analyzed in the Morris water maze (MWM) four days or three months after the soman challenge. Fifty percent of the saline-injected animals that were challenged with soman survived with mild-to-moderate signs of acute toxicity that subsided within a few hours. These animals showed no learning impairment and no memory retention deficit, when training in the MWM started four days post-soman challenge. In contrast, animals presented significant learning impairment when testing started three months post-challenge. Though the magnitude of the impairment correlated with the severity of the acute toxicity, animals that presented no or only mild signs of toxicity were also learning impaired. All guinea pigs that were treated with galantamine survived the soman challenge with no signs of acute toxicity and learned the MWM task as control animals, regardless of when testing began. Galantamine also prevented memory extinction in both saline- and soman-challenged animals. In conclusion, learning impairment develops months after a single exposure to 1xLD50 soman, and galantamine prevents both the acute toxicity and the delayed cognitive deficits triggered by this OP poison.

Figure 1. Flow chart of the treatments and behavioral testing the guinea pigs were subjected to and schematic representation of the zones in the water maze

A. Flow chart showing the times the animals were injected with saline, soman, and/or galantamine and the times at which they were subjected to the different tests in the MWM. B. Diagram shows that, for purposes of analyses, the water maze was virtually divided into four quadrants and a 15-cm wide zone along the wall of the maze. The inner doted circles in the centers of non-target quadrants represent areas of hypothetical platforms and the outer doted circles represent the annulus-40. The filled circle in the target (training) quadrant represents the target platform and the outer circle represents the annulus-40 in the target quadrant.

Figure 2. Acquisition of the MWM task starting four days after injection of guinea pigs with saline, soman and/or galantamine

Experimental groups consisted of animals that were injected with: saline (0.5 ml/kg, im)/saline (0.5 ml/kg, sc), galantamine (8 mg/kg, im)/saline (0.5 ml/kg, sc), saline (0.5 ml/kg, im)/soman (1xLD50, sc), or galantamine (8 mg/kg, im)/soman (1xLD50, sc). Inter-injection intervals were 30 min. Four days after the injections, animals received five days (four trials/day) of reference memory training, followed three weeks later by training to find the hidden platform positioned in the quadrant opposite to the training quadrant. Graphs of escape latency (A) and distance traveled (B) per training day revealed no significant difference among the test groups during the acquisition phase and the training to new platform position. Data points and error bars represent mean and SEM, respectively, of results obtained from 10–15 animals/group.

Figure 3. Peformance of guinea pigs in probe tests performed one day and three weeks after completion of the acquisition phase of the MWM training started four days following the injections of saline, soman, and/or galantamine

Results are from the same animals as those that completed the MWM training shown in Figure 2. A. Data are from the probe test performed 24 h after completion of the acquisition phase. Top graph shows the time animals spent in each of the four quadrants of the pool. Bottom graph shows number of crossings of the platform areas virtually positioned in the center of each quadrant of the pool. Within each experimental group, animals showed bias to the target quadrant and to the platform area in the center of the training quadrant (* p < 0.05 according to one-way ANOVA followed by Dunnett post-hoc test). B. Data are from the probe test performed three weeks after completion of the acquisition phase. Top graph depicts the time spent in each quadrant of the pool. Middle graph shows the number of crossings of the platform areas virtually positioned in the center of each quadrant. Bottom graph shows the distance travelled in the pre-defined 40-cm annulus surrounding the center of each quadrant. Within each experimental group, animals showed no bias to the target quadrant. However, animals pre-treated with galantamine showed preference to crossing the platform and the annulus-40 area vitually positioned in the center of the training quadrant (* p < 0.05 according to one-way ANOVA followed by Dunnett post-hoc test). In addition, galantamine-pretreated animals showed significantly better performance than saline-pretreated expressed as a higher number of the target platform crossings and larger distance traveled in the target annulus-40 zone (# p < 0.05 vs. saline/saline, † p < 0.05 vs. saline/soman according to one-way ANOVA followed by Fisher’s LSD post-hoc test). In A and B, the training (target) area was used as the reference against which the other three areas were named as shown in Figure 1. Graph and error bars represent mean and SEM, respectively, of results obtained from 10–15 animals. The horizontal dotted lines in the top graphs represent the chance level (22.5 s).

Figure 4. Acquisition of the MWM task starting three months after injection of guinea pigs with saline, soman and/or galantamine

Female prepubertal guinea pigs were injected intramuscularly with saline (0.5 ml/kg) or galantamine (8 mg/kg) 30 min prior to the sc injection of 1xLD50 soman (26 µg/kg) or saline (0.5 ml/kg). Three months later, animals received six days (four trials/day) of reference memory training. Graphs of escape latency (A) and distance traveled (B) per training day revealed that saline/soman-injected animals showed significant learning impairment. Galantamine/saline-injected animals also presented a delay in their ability to learn to find the hidden platform; on the second day of training, their escape latency was still longer than that of control (saline/saline-injected) animals. Data points and error bars represent mean and SEM, respectively, of results obtained from 7–11 animals/group. * p < 0.05 vs. saline/saline according to one-way ANOVA followed by Bonfferoni test.

Figure 5. Thigmotactic behavior during the acquisition phase of the MWM initiated three months after injection of guinea pigs with saline, soman and/or galantamine

Results presented here are from the same animals as those in Figure 4. Thigmotactic behavior was defined by the time the animals spent swimming close to the wall (A) and the distance they swum close to the wall zone (B). Data points and error bars represent mean and SEM, respectively, of results obtained from 7–11 animals per treatment group. * p < 0.05 according to one-way ANOVA followed by Fisher’s LSD post-hoc test.

Figure 6. Peformance of guinea pigs in probe tests performed one day and three weeks after completion of the acquisition phase of the MWM training initiated three months following the injections of saline, soman, and/or galantamine

Results are from the same animals as those that completed the MWM training shown in Figure 4. A and B show data from the probe test performed 24 h and three weeks, respectively, after completion of the acquisition phase. Top graphs show the time animals spent in the each of the four quadrants of the pool. Bottom graphs shown the number of crossings of the platform areas virtually positioned in the center of each quadrant of the pool. The training (target) quadrant was used as the reference against which the other three quadrants were named. In A, galantamine/soman-injected animals showed bias to the target quadrant and to the platform area in the center of the training quadrant (* p < 0.05 according to one-way ANOVA followed by Dunnett post-hoc test). In B, galantamine/soman-injected animals showed preference to crossing the target platform area in comparison with the platform area virtually positioned in the opposite quadrant (* p < 0.05 according to one-way ANOVA followed by Dunnett post-hoc test). Graph and error bars represent mean and SEM, respectively, of results obtained from 7–9 animals. The horizontal dotted lines in the top graphs represent the chance level (22.5 s).

Figure 7. Performance of guinea pigs to find relocated platforms following the acquisition phase of the MWM training initiated three months after injections of saline, soman and/or galantamine

Results are from the same animals that completed the second probe test after the MWM training initiated three months after the injections. Each day animals were trained to find the platform in a different position as described in Materials and Methods. They received four trials per day. There were no significant differences among the experimental groups. Data points and error bars represent mean and SEM of results obtained from 7–9 animals/group.

Figure 8. Relationship between scores of acute toxicity and mean escape latency

A. Plot of mean escape latency vs. score of acute toxicity. The escape latency across five or six days of training was averaged for each animal. Lines through the data points are the linear regressions. The Pearson correlation coefficients (r²) of the linear regressions of data from animals tested four days and three months after their injection with saline/soman were 0.14 and 0.51, respectively. The correlation between the severity of the acute toxicity and the learning impairment the animals presented three months after their injection with saline/soman was significant (F = 9.35, p = 0.014). The higher the toxicity score was, the longer the mean escape latency was. B. When tested three months after their treatments, mean escape latency of saline/saline-injected animals was significantly shorter than that of saline/soman-injected animals that showed either no or only mild signs of acute toxicity (scores 0–1). * p < 0.05 according to one-way ANOVA. Graph and error bars are mean and SEM of results obtained from 8–11 animals.

Figure 9. Fluro Jade-B staining of different regions of the brains of guinea pigs treated with galantamine or saline and subsequently challenged with 1xLD50 soman

Guinea pigs were injected with saline (0.5 ml/kg, im) or galantamine (8 mg/kg, im) and thirty min later with soman (1xLD50, sc). Saline/soman-injected guinea pigs classified as mildly to moderately intoxicated and all galantamine/soman-injected guinea pigs were euthanized 48 h after the treatments. Their brains were processed for Fluoro Jade-B staining. Photomicrographs are representative of the pyriform cortex, amygadala, CA1 field of the hippocampus, and striatum of a saline/soman-injected guinea pig that scored 2 in the modified Racine scale (A) and of a galantamine/soman-injected animal (B). No Fluoro Jade-B-positive cells were seen in the brains of galantamine/soman-injected guinea pigs. Results are representative of each treatment group, which had four animals. Calibration bar: 50 µm.