Based on the additive effect of combination therapy of donepezil and regarding the similar neuronal mechanisms through them donepezil and EE exert their enhancing effects on cognition, we hypothesized that an intervention paradigm combining donepezil administration as an AChEI with cognitive stimulation in AD rats would yield greater cognitive benefits than single-domain interventions. AD animals showed impaired function in spatial memory parameters as marked by decreased time, distance, and crossing in the target quadrant in MWM. Pretreatment with donepezil and EE separately could only lead to a slight enhancement in memory parameters. When animals were treated with donepezil and EE simultaneously, they exhibited significantly restored function as seen in saline-treated rats. Hippocampal BDNF expression was decreased in AD animals and combination therapy of donepezil and EE could reverse it to the control levels.
Alzheimer's disease is characterized by the progressive accumulation of senile plaques and neurofibrillary tangles in the brain, which are associated with neuronal damage, particularly in the cholinergic system. The amyloid-beta peptide is the main component of senile plaques, and it is known as a causal factor in the development and progress of AD (Nelson et al., 2009). Animal models with AD exhibit progressive and long-term deficits in memory function which are similar to the symptoms of sporadic AD (Facchinetti et al., 2018; LaFerla & Green, 2012). Our data confirmed the previous studies showing that ICV or intrahippocampal injection of amyloid-beta causes memory impairments. However, a single injection of amyloid peptide does not express all of the pathological features of AD. There is no robust animal model that reproduces all of the characteristics of the disease, and genetic mouse models, as well as ICV or intrahippocampal injection of amyloid proteins, are the most widely applied methods for experimentally induced AD (Facchinetti et al., 2018). The injection of amyloid-beta into the rat brain induces pro-inflammatory reactivity, oxidative stress, and a cascade of neurotoxicity that ultimately leads to the loss of neuronal functions involved in the behavioral symptom of AD (McLarnon, 2014). Therefore, the injection of amyloid-beta is an alternative AD animal model to transgenic animals.
Both donepezil and EE have been shown to improve cognitive ability scores in healthy experimental subjects as well as in experimental models with cognitive impairments (Berardi et al., 2007; H. B. Guo et al., 2015; T. S. Lee et al., 2013). However, these paradigms in the present study could not separately lead to a significant recovery of the impaired memory of AD animals. Among the several probable factors, the dose of donepezil used and the time course effect of EE exposure in this study may be the most important ones. By this assumption, Cavalcante et al. (Cavalcante, 2019) reported that only short-term (two weeks) but not long-term (four weeks) exposure to an EE could promote the extinction of aversive memory. The cognitive benefits of donepezil administration in AD patients are widely suggested to be dose-response related (Jelic & Darreh-Shori, 2010; J. H. Lee et al., 2015). In this study, we orally administrated a 4 mg/kg dose of donepezil once a day for 3 weeks. The administered dose of donepezil in animal studies varies between 0.5 and 10 mg/kg. The approved standard doses of 5 and 10 mg/day inhibits cortical AChE activity by only 20–40%, but based on the dose-response relationship with donepezil, it was expected that increasing dosage to 23 mg/day further increases AChE inhibition (Cummings et al., 2013; Farlow et al., 2010). The dose-dependent effect of donepezil administration on the cholinergic system is also reported in animal studies (Chamoun et al., 2016; Kasa et al., 2000). Thus, the dose used in the present study may not be enough to produce separately significant improvement in the memory function of AD animals.
The primary goal of this study was to test whether co-exposure to donepezil and EE is more efficient in improving cognitive deficits in AD rats than either paradigm alone. Results showed that while neither EE nor donepezil alone could significantly restore spatial memory scores in AD rats, combination therapy was effective. It seems from the inspection of the data that the combination therapy was not synergistic but may be additive, with each paradigm alone producing some improvement which when combined reached significance. All individual groups performed the learning trials successfully; however, while the Alzheimer group showed a considerable increase in the acquisition blocks, it did not meet the significant level. Given this, and since the swimming speed was the same among the groups, it could be assumed that the observed differences in memory function may not be related to confounding factors such as specific differences in sensorimotor integration or motivation of the animals.
Donepezil in combination with a variety of agents such as piperine (Nazifi et al., 2021), resveratrol (Rao et al., 2021), estradiol (Gibbs et al., 2011), and manual acupuncture (Jiang et al., 2019) has been examined to achieve a superior benefit for neurobehavioral deficits in AD animal models. To date, there have been no attempts to address the potentials of donepezil administration and EE in combination for the prevention of cognitive deficits in AD patients or experimentally neurodegenerative status. The partially analog studies are those that aimed to evaluate the combined benefit of cognitive rehabilitation plus donepezil for AD patients (Giordano et al., 2010; Matsuzono et al., 2016). One found that the combination of cognitive rehabilitation plus a choline esterase inhibitor donepezil resulted in a better effect on the Mini-Mental State Examination test scores in AD patients than only drug therapy (Matsuzono et al., 2016). These findings suggest that the combination pattern of donepezil with cognitive stimulation programs is more efficacious than AChEI alone for improving cognitive and behavioral deficits in the case of CNS insults and neurodegenerative staus.
Next, we sought the hippocampal expression of BDNF which is a key signaling molecule in memory formation and AD neurodegeneration (Chen et al., 2017; Cunha et al., 2010). It has been reported that donepezil increases the serum levels of BDNF in patients with AD, and BDNF upregulation is involved in the protective effect of AChEI (Leyhe et al., 2008). The contribution of nerve growth factors to the enhancing effect of EE on neuronal functioning has also been well-documented (Rossi et al., 2006). Therefore, we examined the potential involvement of hippocampal BDNF in memory status by measuring its mRNA expression in animals. We found that BDNF expression was suppressed in AD rats and pretreatment with donepezil plus EE could increase it to the saline levels. These results are consistent with the previous findings showing that a suppressed hippocampal BDNF in the amyloid-beta model of AD rats (Kim et al., 2014). Thus, the observed additive outcome of donepezil and EE combination for memory performances of AD rats may be in part due to the augmented influence of this combination therapy on BDNFexpression. A human study by Alvarez et al. (Alvarez et al., 2016) evaluated the effects of Cerebrolysin, donepezil, and combined therapy on BDNF serum levels and cognition at week 16 and week 28 after treatment mild-to-moderate AD patients. Cerebrolysin, but not donepezil, increased serum BDNF at week 16, while the combination therapy enhanced it at both time points. The increasing effect of combination therapy was significantly more profound than donepezil and Cerebrolysin groups at week 16 and week 28, respectively. These findings were associated with better cognitive improvements in AD patients, suggesting an advantage of combination therapy again for achieving the most benefits from medication in AD.
Results of AchM1R expression within the hippocampus revealed that while there was no significant difference among the groups, the AD group showed an increase in AchM1R levels, and don, EE, and especially don+EE diminished it toward the control levels. Intracerebral injection of amyloid-beta leads to the loss of cholinergic neurons and decreased Ach levels (Giovannini et al., 2002). Therefore, the observed pattern of modulations in AchM1R expression can be assumed to be related to the balance between ligand concentration and receptor expression.