The establishment of preeclampsia-like rat models
To confirm whether the preeclampsia animal model was successfully established, blood pressure and 24h urine protein of pregnant rat was examined. We found the administration of L-NAME led to an increase in systolic blood pressure. Before the administration of L-NAME, the average systolic blood pressure was 126.20 mmHg. However, the average SBP was as high as 146.53 mmHg after using it (Figure 2a, p<0.001). Meanwhile, it also led to an significant increase in urine protein level in pregnant rat (Figure 2b, p<0.001). In addition, the body weight of neonates in the preeclampsia group was significantly lower (5.76±0.63 g) than that of the control group (7.72±0.85 g) (Figure 2c, p<0.001). The number of neonates in preeclampsia group was also decreased compared with the control group (Figure 2d, p<0.05). Furthermore, there was limb defect in some neonates of preeclampsia group (Figure 2e, p>0.05).
Improvement in learning and memory ability in the offspring of L-NAME group after EE
To analyze the learning ability and memory of the offspring, Morris water maze was used. In both stage, the performance of offspring in EE group was indistinguishable from that of offspring in control group- in sharp contrast to offspring in L-NAME group.
In the training stage, the latency to platform was progressively decreased in control group (Figure 3a), which indicated that offspring learned the task from day1 to day4. However, latency to platform in L-NAME group decreased much slower, less efficient and longer than that in control group (Figure 3a, p<0.005). While EE could largely restore the learning ability deficient, reflected by the decrease in latency to platform (Figure 3a, p<0.005). There is no significant difference in swimming speed among 3 groups (Figure 3b, p>0.05). The performance in the training stage revealed that offspring in L-NAME group had impaired learning abilities and EE could improve it.
At the memory test stage, the results showed that offspring in L-NAME group displayed much higher latency to training platform area, compared with control group(Figure 4a, p<0.005). A shorter swimming distance in target quadrant was found L-NAME group (Figure 4b, p<0.005). They also spent significantly less time in platform quadrant than offspring in control group (Figure 4c, p<0.005). Additionally, we found the frequency of crossing platform in L-NAME group was much lower than that in control group (Figure 4d, p<0.001). All these changes could be reversed by EE. Therefore, we have demonstrated that EE had protected offspring of PE from impaired memory.
Increased neurogenesis in hippocampus of offspring of preeclampsia after EE
To reveal cellular and molecular mechanism through which EE improves brain function in the offspring of preeclamptic rat, we investigated the hippocampal neurogenesis, which was reported to be associated with spatial learning ability and memory. Immunofluorescence (IF) results showed that numbers of BrdU+ cells was significantly decreased in L-NAME group, while it was increased in EE group (Figure 5a,5b, p<0.005), indicating that decreased neurogenesis was resolved by EE. Next, qRT-PCR was used to test the expression level of adult hippocampal neurogenesis –associated genes including Fgf, PTN, EP300, Creb, BNDF and NGF. However, there were no significant difference in expression levels of these genes among three groups (Figure 5c, p>0.05). We then tested whether there was changes in VEGF concentration. VEGF was reported to promote neurogenesis in adult brain possibly through enhancement in vascular niche. The result showed a significant reduction of VEGF in L-NAME group when compared with young group. However, EE restored hippocampal VEGF to a level similar to those of control group (Figure 5d, p<0.05). Thus, EE prevent hippocampal VEGF decrease in L-NAME offspring, possibly underlying its positive effect on neurogenesis.
Increased synaptic plasticity in hippocampus of the offspring of the L-NAME group after EE
To evaluate the synaptic plasticity in hippocampus, we assessed the mRNA expression level of pre- and postsynaptic proteins synapsin, PSD95 and SNAP25. We found decreased expression levels of synapsin (Figure 6a, Control vs L-NAME, p<0.005, L-NAME vs EE, p<0.05) and PSD95 in L-NAME group (Figure 6b, p<0.05) compared with control group. However, the expression level of these synaptic-related protein was restored by EE. No significant difference in SNAP25 level was observed among these groups (Figure 6c, p>0.05).
Reduced apoptosis in hippocampus of the offspring of the L-NAME group after EE
Neural apoptosis in DG region of hippocampus, measured by the staining of TUNEL+ nuclei, was significantly different among three groups (Figure 7a). The number of TUNEL+ cells were increased in L-NAME group when compared with control group as well as EE group. No significant difference existed between control and EE group (Figure 7b, p<0.001). The results indicated that EE could alleviated neural apoptosis in L-NAME group.
Reduced inflammation in hippocampus of offspring of preeclampsia after EE
Considering the strong association between cognitive impairment and inflammation, we examine the inflammatory cytokines in hippocampus. Both qRT-PCR and ELISA results revealed different inflammatory profiles in these groups. The results showed both mRNA and protein levels of pro-inflammatory cytokines including IL-1β (Figure 8a, Control vs L-NAME, p<0.005, L-NAME vs EE, p<0.05. Figure 8b, Control vs L-NAME, p<0.001, L-NAME vs EE, p<0.05 ) and IL-6 were increased in L-NAME group compared with control and EE group (Figure 8c, Control vs L-NAME, p<0.001, L-NAME vs EE, p<0.005. Figure 8d, Control vs L-NAME, p<0.005, L-NAME vs EE, p<0.05). Nonetheless, no significant difference was found in TNF-α level among three groups (Figure 8e,f, p>0.05). These results suggested that EE could reverse excessive hippocampal inflammation in L-NAME offspring.