Mice behavioral characteristics after pilocarpine injection
Mice often exhibited hypoactivity, curling up, tremors, head bobbing, and myoclonic movement of the limbs after pilocarpine injection. SE usually began by rearing with or without falling and jumping at the onset of SE.
Saracatinib reduces the frequency of epileptic spikes after SE.
Saracatinib, as a pharmacological inhibitor of the Fyn, is effective at suppressing epileptiform spikes . We therefore examined the effects of saracatinib on the frequency of spikes using our pilocarpine induced SE model. We found that post-SE saracatinib injection decreased the frequency of epileptic spikes in the hippocampus (Fig. 2b) ( t(8) = 2.986, p = 0.007). Furthermore, video-EEG showed that saracatinib injection decreased the duration (Fig. 2c) ( t(8) = 2.763, p = 0.025) and frequency (Fig. 2d) ( t(8) = 3.715, p = 0.006) of spontaneous seizure after SE (Table 1). These results suggested that our protocol was successful at inducing SE, and that saracatinib injection was effective at relieving SE.
Saracatinib inhibits cognitive function deficits after SE.
Cognitive function deficits are a common sequelae after SE, so we examined whether saracatinib inhibited deficits in cognitive function after SE. Using the MWM test, escape latency (Fig. 3a) (F(2,22) = 3.68; CON vs SE: 33.59 + 3.8 vs 50.29 + 4.87 on 3 days, p = 0.13; F(2,22) = 7.116; CON vs SE vs Sar + SE: 22.13 + 3.4 vs 45.83 + 5.8 vs 30.11 + 4.18 on 4 days, p = 0.01 in CON vs SE, p = 0.025 in SE vs Sar + SE; F(2,22) = 10.307; CON vs SE vs Sar + SE: 17.42 + 2.88 vs 41.72 + 3.94 vs 26.45 + 4.69 on 5 days, p = 0.009 in CON vs SE; p = 0.012 in SE vs Sar + SE) and swimming path length from the starting position to the target platform (Fig. 3b) (F(2,22) = 3.02; CON vs SE: 1589.37 + 213.03 vs 2490.87 + 296.99 on 3 days, p = 0.23; F(2,22) = 8.54; CON vs SE: 1138.7 + 166.62 vs 2381.65 + 258.46 on 4 days, p = 0.15; F(2,22) = 13.81; CON vs SE vs Sar + SE: 802.907 + 103.21 vs 1786.48 + 155.98 vs 1070.8637 + 113.52 on 5 days, p = 0.001 CON vs SE; p = 0.16 SE vs Sar + SE ) were decreased in Sar + SE mice, when compared with the SE mice. However, the swimming speed was similar in all groups mice (Fig. 3c) (F(2,22) = 0.54; CON vs SE vs Sar + SE: 62.09 + 2.69 vs 61.72 + 3.02 vs 57.85 + 3.79 on 3 days, p > 0.05; F(2,22) = 0.475, CON vs SE vs Sar + SE: 57.46 + 2.87 vs 55.94 + 3.3 vs 59.62 + 2.6 on 4 days, p > 0.05; F(2,22) = 0.11, CON vs SE vs Sar + SE: 59.01 + 2.36 vs 57.85 + 2.7 vs 57.43 + 2.46 on 5 days, p > 0.05). There results suggested that decreased escape latency and swimming path length in the Sar + SE group mice were not due to an impaired capacity of swim. When the target platform was removed in the probe trails, the number of crossing target platform areas (Fig. 3d) (F(2,22) = 5.917; CON vs SE vs Sar + SE: 3.22 + 0.43 vs 1.25 + 0.25 vs 2.5 + 0.5, p = 0.012 in CON vs SE; p = 0.04 in SE vs Sar + SE) and staying time in the quadrant (Fig. 3e) (F(2,22) = 5.202; CON vs SE vs Sar + SE: 20.8 + 1.97 vs 12.84 + 2.14 vs 18.237 + 0.922, p = 0.01 in CON vs SE; p = 0.38 in SE vs Sar + SE) where the target platform was previously located were increased in the Sar + SE group, when compared with the SE group. Furthermore, when we performed the NOR test, the PI was similar in both SE group mice and Sar + SE group mice (Fig. 3f) (F(2,25) = 0.326; CON vs SE vs Sar + SE: 0.58 + 0.017 vs 0.553 + 0.02 vs 0.545 + 0.049, p > 0.05), but the RI of the Sar + SE mice was increased (Fig. 3g) (F(2,25) = 6.944; CON vs SE vs Sar + SE: 0.64 + 0.029 vs 0.453 + 0.039 vs 0.578 + 0.036, p = 0.015 in CON vs SE; p = 0.023 in SE vs Sar + SE). Together, these results showed that saracatinib inhibited deficits in cognitive function after SE.
Saracatinib inhibits the loss of neurons in the hippocampus after SE
Because hippocampal neuron loss is an important reason for the deficits in cognitive function after SE, we next determined the number of hippocampal neurons. Neurons in the DG region in the hippocampus were visualized by anti-NeuN antibody using immunohistochemical staining (Fig. 4a). The results showed that the number of neurons was increased in the DG in the hippocampus after saracatinib treatment than the SE groups (Fig. 4b) (F(2,12) = 12.32; CON vs SE vs Sar + SE: 30.32 + 1.65 vs 20.48 + 1.09 vs 26.44 + 1.42, p = 0.005 in CON vs SE; p = 0.032 in SE vs Sar + SE), suggesting that saracatinib relieved the cognitive function deficits by rescuing hippocampal neuronal loss during SE.