In this study, we examined the effects of an EE intervention in early life for the prevention of preeclampsia-related cognitive decline in adolescent offspring in an L-NAME-treated rat model. Previous epidemiological studies have demonstrated that maternal preeclampsia is strongly associated with poor cognitive performance of their children. Consistent with this, we found that the administration of L-NAME to pregnant rats induced cognitive deficits in offspring. Offspring in the L-NAME group exhibited pathological changes, including impaired neurogenesis and synaptic plasticity, increased neural apoptosis and increased levels of inflammatory cytokines in the hippocampus compared with their counterparts in the control group. Notably, rearing offspring in the L-NAME group in an enriched environment for five weeks prevented hippocampus-dependent learning ability and spatial memory decline, NOR deficits as well as pathological changes in the hippocampus. Altogether, these results indicated that EE might effectively reverse cognitive changes caused by an adverse intrauterine environment.
We used an L-NAME rat model of preeclampsia, in which L-NAME (an inhibitor of NOS) was administered to pregnant rats during gestational days 13 to 21. This model is effective in exploring preventive strategies for cognitive decline in offspring who were born after preeclampsia due to its ability to recapitulate the clinical features of preeclampsia, including increased blood pressure and urine protein. In women with preeclampsia, NO production is reduced. The L-NAME rat model could mimic the NO deficiency. Moreover, since gestational days 13 to 21 are a critical stage for brain development, this model is effective in studying the neurodevelopment of preeclampsia offspring. A previous study from our laboratory reported that the spatial learning ability and general learning ability decline and there is impaired neurological development among adolescent offspring of L-NAME rats, which supports the effectiveness of this model.
Few studies have explored ways to improve the cognitive ability of preeclampsia offspring and they have mainly focused on gestational diet interventions. To our knowledge, the present study represents the first examination of whether EE could protect preeclampsia offspring against cognitive decline and thus provides novel insights for early intervention. We showed that EE in early life was sufficient to prevent cognitive deficits in adolescent offspring from the L-NAME group. There are various ways to provide an enriched environment; thus, the protocols lack consistency. However, the most common procedure includes rearing the rats in a large cage and providing them with novel subjects and social contact for at least 30 days starting immediately after weaning. This procedure provides all of the key factors of EE, including social contacts, novelty and exercise, all of which have been reported to be rewarding. The offspring were weaned at postnatal day 21. Therefore, they were reared in an enriched environment from postnatal day 21 to day 56, which lasted for five weeks.
In this study, the Morris water maze and NOR task were used to assess cognitive function in offspring. Our data showed that offspring in the L-NAME group exhibited a clear decline in spatial learning ability, reflected by an increased “latency to platform”, which was prevented by EE. With regard to spatial memory, L-NAME induced a spatial memory decline reflected by a shorter swimming distance, less time in the target quadrant and a lower “frequency of crossing the platform” in the test stage. Impaired spatial memory was also revolved by EE. These results showed impaired hippocampus-dependent learning ability and spatial memory in the L-NAME group. However, after 5 weeks of the EE intervention, the performance of offspring in the Morris water maze was dramatically improved. Moreover, offspring in L-NAME group showed an impaired NOR performance which were reflected by the reduced Discrimination index. Five weeks of EE could prevent NOR deficits, the DI of which was similar to the control group.
In an attempt to discern the biological underpinnings of the observed cognitive changes, we focused on the structural and molecular plasticity of the hippocampus. The results showed that EE could improve neurogenesis, attenuate neural apoptosis, and improve synaptic plasticity. Moreover, EE normalized the inflammatory balance in the hippocampus by decreasing the expression of the proinflammatory cytokines IL-1β and IL-6.
The hippocampus is a key structure involved in learning and memory. The adult hippocampus can continuously generate new neurons that are integrated into hippocampal circuits. These newly generated neurons are thought to play an important role in hippocampal-dependent spatial learning and memory (BruelJungerman et al., 2007; Shors et al., 2001). The process of hippocampal neurogenesis has been reported to be influenced by various factors, including physiological conditions and environmental stimuli. Therefore, we investigated neurogenesis in the DG, where new neurons are added to the mature circuit. Our study showed that exposure to an adverse uterine environment exerted a negative effect on hippocampal neurogenesis, reflected by a decreased number of BrdU + cells in the DG region of the hippocampus of offspring, while five weeks of EE intervention in early life could alleviate these changes. This suggests that impaired cognitive function in L-NAME offspring may be associated with reduced neurogenesis, which is attenuated by EE.
Alterations in hippocampal growth factors might be functionally linked with neurogenesis changes. BDNF has been widely studied as a candidate mediator of changes in hippocampal neurogenesis induced by environmental stimuli. For instance, the deletion of TrkB (BDNF receptor) reduced the effects of exercise on adult neurogenesis. In addition, NGF impacts the survival of neuronal progenitor cells. Amy et al. found that cognitive decline with aging was associated with a reduction in NGF(Birch and Kelly, 2019). Moreover, FGF signaling pathways play a role in regulating neurogenesis. The deletion of FGF receptor genes could result in a dramatic loss of neurogenesis in the DG, while enhancing FGF receptor activity in neurogenic cells could increase their numbers(Kang and Hébert, 2015).
VEGF has also been reported to enhance neurogenesis(Fabel et al., 2003; Gao et al., 2009). Gao et al. reported that reduced VEGF expression with aging might exert an impact on the angiogenic niche within the DG and reduce neurogenesis(Gao et al., 2009). VEGF could enhance the angiogenic niche in the subgranular zone (SGZ) of the DG. In support of these studies, blockade of VEGF eliminates exercise-induced improvements in neurogenesis, indicating that VEGF may play a significant role in the stimulation of neurogenesis(Fabel et al., 2003).
These studies indicate the essential roles of these growth factors in the regulation of neurogenesis. Accompanying the changes in neurogenesis, we found decreased expression levels of VEGF, while there were no changes in BDNF, NGF, FGF or other neurogenesis-related genes. Interestingly, the reduction in VEGF was attenuated in the EE group. Furthermore, an increased neural apoptosis level was observed in the L-NAME group, which was attenuated by EE. Therefore, the improved neurogenesis mediated by EE may be partly attributed to increased expression of VEGF and the inhibition of apoptosis.
Previous studies have shown a substantial link between neuropsychological disorders and neuroinflammation. McAffose et al. demonstrated that elevated or prolonged exposure to inflammatory mediators could have detrimental effects on cognitive function. Moreover, recent evidence has implicated dysregulated inflammation in the development of ASD(Matta et al., 2019). Masi et al. reported that a number of cytokines were dysregulated in ASD and were correlated with the severity of the ASD symptoms(Masi et al., 2017). Similar to these findings, in this study, we found increased inflammatory cytokines in preeclampsia offspring compared with normal pregnant offspring. The data in our study indicated a proinflammatory phenotype in the hippocampus of offspring in the L-NAME group, reflected by increased levels of IL-1β and IL-6, which was attenuated by EE. IL-1β has been widely reported to have memory-modulating effects and it is increased in many neurodegenerative diseases and normal aging(Frank et al., 2006; Lynch, 2010); thus, the EE-induced modulation of IL-1 expression could help preserve cognitive function. Additionally, EE-induced improvement in cognitive ability could be partly attributed to the attenuation of the increase in IL-6, which could impact synaptic plasticity and neurodegeneration. Indeed, in this study, we found decreased expression levels of the pre- and postsynaptic proteins synapsin, PSD95 and SNAP25 in the L-NAME group compared with the control group, while EE rescued this reduction. Notably, recent studies have demonstrated that neuroinflammation is a potent inhibitor of hippocampal neurogenesis. Thus, we speculated that reduced inflammation induced by EE attenuated cognitive impairment by improving hippocampal neurogenesis and synaptic plasticity.