Materials and Methods
Animals and treatments
Sprague-Dawley pregnant rats (total of 24, 6 in each group) (Harlan Sprague Dawley, Inc, Chicago, IL) were obtained at gestational day 11 (term = 21 days) and allowed to acclimate for 7 days prior to initiation of experiments. Animals were maintained at controlled temperature (37°C) and controlled light (6:00 am , lights on; 6:00 pm , lights off) as well as controlled facilities with access to food and water ad libitum throughout the study. Saline and MG were administered subcutaneously and LPS was administered intraperitoneally. LPS ( Escherichia coli , serotype 0111:B4) was reconstituted in physiological saline and administered at 500 μg/kg of birthweight.
At gestational day 18, 24 rats (6 in each group) received injections of i.p. LPS (500 μg/kg) or saline of equivalent volume at time 0. Dams were randomized to treatment with subcutaneous normal saline (NS) or MG (270 mg/kg loading followed by 27 mg/kg every 20 minutes) for 2 hours prior to and MG or saline for 2 hours following the i.p. LPS or saline (total 10 doses of 27 mg/kg) and another injection of MG 270 mg/kg at 2 hours following the LPS or saline injection. This resulted in four study groups: (control: NS-NS-NS; LPS: NS-LPS-NS; MG: MG-NS-MG; LPS-MG: MG-LPS-MG).
Pups were delivered spontaneously at term (embryonic day 21). Litter size was maintained at 6-8 pups (with equal numbers of males and females when possible) to ensure equal access to maternal nutrition across litters. The males and females remained with the dams during lactation. At 1 and 3 months of life, at least 11 male pups (≥1 pup from each dam, selected randomly) of each of the groups underwent the 2-way shuttle avoidance protocol (discussed in the following text). We used the same males for the 1-month and 3-month experiments. The numbers of males was 11, 12, 12, and 12 for the LPS, MG, LPS-MG, and control groups, respectively, at 1 and 3 months of age. In this experiment we used only male offspring to overcome any sex differences between males and females.
Two-way shuttle avoidance protocol
To study the rats learning and memory abilities, we used a 2-way shuttle avoidance box. The 2-way shuttle avoidance box, placed in a dimly lit, ventilated, sound-attenuated cupboard, is a rectangular chamber (60 × 26 × 28 cm) divided by an opaque partition with a small passage (10 × 8 cm) connecting 2 equal sized, side-by-side, cube-shaped compartments. Both compartments’ metal grid floors are weight sensitive; microswitches transmit information on the rat’s location to a computer-controlled and automated data collection program managing both the conditioned stimulus (CS) presentations (a tone produced by loudspeakers located on the distal walls of the compartments) and the unconditioned stimulus (US) presentations (electric shock deliveries) as well as recording the rats’ responses (adapted from the report by Tsoory and Richter-Levin, 2006).
Procedure
Each rat completed 1 session of 70 trials of trace conditioning. Each session is comprised of 3 parts: the CS, the US, and the intertrial interval (ITI). These included the following: CS, 10 seconds of tone presentation; US, immediately following the termination of the CS, an electric shock (0.8 mA) is delivered for a maximum of 10 seconds; and ITI, 30 ± 6 seconds. The ITI is the time that elapses between the previous trial and the next. The duration varies, keeping the rat from predicting the arrival of the next tone.
Rats produce 1 of 3 responses: (1) avoidance, which includes shuttling to the adjacent compartment upon hearing the CS tone, the tone stops and an ITI starts, and the rat avoids the electric shock; (2) escape, which includes shuttling to the adjacent compartment while the shock is on, and the shock stops and an ITI starts; or (3) no response, which includes failing to move to the adjacent compartment, and the ITI commences at the end of the 10 second shock. Thus, the rat is subjected to the full duration of the electric shock. The avoidance/escape scale is the average number of avoidances/escapes per block per group.
The primary outcome is the measure of learning and memory abilities. The variable studied is the ratio of avoidances/escapes to the total number of trials as analyzed in blocks of 10 trials each. In the model, the ability of a rat to avoid an electric shock demonstrates higher learning and memory abilities.
Statistical analysis
Statistical analysis was performed using SPSS version 21 (SPSS Inc, Chicago, IL). The normality of the data was tested by the Kolmogorov-Smirnov test. Because some of the parameters were not normally distributed, the Mann-Whitney U test and the Kruskal-Wallis with multiple comparisons were used to analyze differences among the 4 treatments groups (LPS, MG, LPS-MG, and control). Related sample Friedman’s 2-way analysis of variance with pair-wise comparisons was used to determine whether there were significant differences among the seven blocks.
The 70 trials of trace conditioning were divided consecutively into 7 blocks comprised of 10 trials. Each block analyzed was comprised of the average avoidance/escape/no-response, of all the pups in each study group. Analysis was performed on the 7 blocks so that, for example, the first 10 trials (first block) are compared with the second 10 trials (second block) or the last 10 trials (seventh block). A value of P < .05 was considered as statistically significant.
Ethical approval
The protocols and procedures were approved by the Institutional Animal Care Committee at the Rappaport Research and Education Institute (Haifa, Israel).
Results
Newborn pups
There was no difference in the weight of the pups in the different groups: 7.2 ± 1.1 g, 6.2 ± 1.1 g, 6.5 ± 0.6 g, and 6.4 ± 0.6 g in the control, LPS, LPS-MG, and MG, respectively. No differences were noted in the male/female ratio. All dams gave birth at term. There was a trend toward fewer newborn pups in the LPS group compared with the control (6 ± 4 vs 11 ± 2; P = .06). There was no significant difference among the other groups: MG, 11 ± 2, and LPS-MG, 7 ± 4.
All rats either escaped or avoided on all trials. None of the rats demonstrated no response, which would imply a priori severe damage. Results were compared among treatment groups, ages, and individual blocks.
Month 1
Avoidance
At 1 month of age, all the groups demonstrated improvement in their learning abilities from the first block to the seventh block ( Figure 1 ). In each group, the average number of avoidance responses was determined, and then the results were averaged. The Kruskall-Wallis test with multiple comparisons was performed to assess the difference in avoidance response among the different groups. When the groups were compared, there was a significant difference between the learning ability of the MG group and the control group. Median (lower-upper quartiles) for avoidance was 2.93 (1.73–4.27) vs 1.29 (0.41–2.29) ( P = .048), respectively ( Figure 2 ), suggesting, that MG may have an enhancing effect on the offspring’s learning abilities. There were no differences among the other treatment groups.
Escape
At 1 month of age, all groups except for the LPS-MG demonstrated a significant increase in the number of escapes between the first block and the seventh block ( Figure 3 ).
Month 3
Avoidance
At 3 months of age, the LPS group did not demonstrate any improvement in learning abilities from the fourth to the seventh block, whereas all the other groups demonstrated a significant improvement in their learning abilities ( Figure 4 ). When an analysis was performed on each group to evaluate the learning curve from one block to the following block, the LPS group demonstrated improvement in a single block, whereas the control, MG, and LPS-MG groups demonstrated significant improvement in learning abilities in 3 blocks ( Figure 4 ).
On the final block (seventh block), the LPS-MG offspring demonstrated the highest learning abilities with the best avoidance scores, significantly higher than all other groups. In a multicomparison test, the avoidance of LPS-MG was significantly higher than in LPS alone. Median (lower-upper quartiles) for avoidance LPS-MG was 9.00 (7.25–10.00) vs LPS 5.00 (0.00–9.00) ( P = .05). When the groups were compared in pairs, LPS-MG was significantly better at avoidance than LPS and controls. Median (lower-upper quartiles) for avoidance was 9.00 (7.25–10.00), 5.00 (0.00–9.00), and 6.00 (2.00–8.75) for LPS-MG, LPS, and controls, respectively ( P < .016) ( Figure 5 ).
When the learning abilities were compared in each group between the matching blocks at 1 and 3 months of age, the control and LPS-MG groups demonstrated significant improvement in 5 of the 7 blocks ( P < .05), whereas the LPS and MG groups demonstrated improvement only in 1 of the 7 blocks ( P < .05). This finding is consistent with learning and memory impairment in the LPS group when compared with the control (saline) and LPS-MG groups ( Figure 6 ).
The MG group had the highest scores at 1 month, which can account for part of the lack of improvement in this group. We further tried to evaluate whether the improvement in the learning abilities in each group between the blocks in the first and third months increases as the blocks progress or whether the improvement is static. We found that the LPS group did not demonstrate any improvement, whereas the control, MG, and LPS-MG groups demonstrated significant improvement ( P < .05).
The greatest improvement in learning abilities was demonstrated in the LPS-MG group, implying that the increase in learning abilities is adaptive and improves in this group not only from 1 month to 3 months but also from one block to the next ( Figure 7 ).
