The purpose of this study is to investigate the protective effect of levosimendan vis-á-vis scopolamine induced cognitive impairment. To confirm this hypothesis, a predominantly used scopolamine model for the screening of drugs to treat Alzheimer’s disease was adopted [36, 49]. Scopolamine, a post-synaptic muscarinic acetylcholine receptor (mAChR) antagonist, down regulates the cholinergic transmission. It leads to decreased acetylcholine release and results in cognitive dysfunction [50]. Further, scopolamine at high doses impairs the cognitive function via activation of BACE-1 enzyme, Amyloid-β accumulation, oxidative stress and neuroinflammation [51].
In this study, open field test was performed to address the self-independent exploring property and general analysis of rats’ locomotor behaviour. As shown in (Fig.-2), there were no change in the parameters analysed among all the groups. In light of this evidence, we can state that scopolamine administration does not impact the locomotor activity and the behavioural deficits observed in NORT, Y maze and water maze were solely due to its impairment of cognitive function [52, 53]. Novel object recognition (NOR) test was carried out to assess the visual recognition of animal and exploration of novel object in a familiar environment. In this test, non-spatial recognition ability termed as discrimination index was measured to evaluate the cognitive performance. In the present study (Fig. 3), we demonstrated that scopolamine injection decreases the discrimination index of the animal. Whereas, levosimendan countered this result and ameliorated the decrease in discrimination index caused by scopolamine and the results are in accordance with the previous studies [54, 55]. On the other hand, Y-maze test is used predominantly to measure the short-term spatial working memory [56]. In this % spontaneous alternation was used as an indicator for short-term working memory [51]. Previous studies reported that scopolamine administration decreases the short-term memory and it is evidenced by decrease in % spontaneous alterations [27, 57]. Results from (Fig. 4) this study were in line with previous studies and further, levosimendan pretreatment protected short-term memory decline [58].
MWM test was performed to measure the deficits of learning and memory associated with hippocampal dysfunction. In this test, non-spatial learning and memory was postulated by gauging the latency time to identify the hidden platform. In this study, scopolamine administered rats were observed with increase in latency time to find the platform when compared with the normal control group. It indicates the impairment in learning and memory. [59]. The animals treated with levosimendan had shown decrease in the escape latency time while comparing with the scopolamine alone treated group, represents its protective ability against cognitive impairment (Fig. − 5A). Further, in the probe trial test, scopolamine administered rats spends less time in target quadrant. However, levosimendan treated rats spent more time in target quadrant comparing to the disease group signifying its role in improving spatial learning and memory. All the results in MWM test were familiar with earlier reported studies (Fig.- 5B) [60]. In this context, the results of behavioural tests suggests that levosimendan treatment could ameliorates long-term and reference memory impairment induced by scopolamine.
The cholinergic neurotransmission is involved in many physiological processes, including synaptic plasticity, learning and memory. Dysfunction of cholinergic neurons in the brain decreases the release of Ach and results in cognitive impairment. Further, cholinergic agonists like donepezil, rivastigmine facilitate the memory and cholinergic antagonists like scopolamine impair the memory [61]. In that sense, molecules that modulate cholinergic hypoactivity by slowing the rate of acetylcholine degradation have potential clinical use. In order to elucidate the effect of levosimendan on cholinergic function, we have estimated the activity of AChE and BuChE [62]. According to the previous studies, scopolamine administration produced severe cholinergic deficits evidenced by increase in AChE and BuChE activity and decreases synaptic plasticity [43, 63]. However, levosimendan pre-treatment significantly decreased the enzyme activity and loss of cholinergic neurons induced by scopolamine (Fig. 6). Our results in this study are relevant to the previous studies [64, 65]. These findings agree with the behavioral data and together suggest that ameliorative effect of levosimendan against scopolamine induced memory impairment.
Furthermore, many devastating pieces of evidence suggest that oxidative stress is another causative factor of Alzheimer pathology [66]. In experimental animals, amnesia induced by scopolamine has been reported to be associated with increase free radicals in the brain, especially hippocampus. In agreement with the previous studies, our study results showed that scopolamine administration resulted in remarkable oxidative damage to the hippocampus and it is evidenced by increased levels of MDA and NO, a valid index of lipid peroxidation and significant decrease in antioxidant defense systems such as GSH and SOD [9, 67]. In contrast, levosimendan administration showed its beneficial role against oxidative stress by increasing the activity of GSH, SOD and by decreasing the lipid peroxidation and No levels when compared to scopolamine alone treated group. All the results from oxidative stress parameters were in line with the previous studies (Fig. 7,8) [67]. These data demonstrated that levosimendan has free radical scavenging activity and increases the levels of anti-oxidants. Hence, it suggested that the reduction of oxidative stress could be related to the cognitive effect of levosimendan in scopolamine treated rats [12, 19, 26].
Microglia, an inhabitant immune cells of the brain, is regularly inspect the microenvironment under biological conditions. In AD, activation of microglia mediated by amyloid-β deposition and oxidative stress initiates cerebral neuroinflammation. Microglial polarization in the brain plays a detrimental role by provoking the expression of pro-inflammatory cytokines. The transcription factor, NF-κB, involved in various gene regulations in inflammation [68]. It has been reported scopolamine injection initiates the production of inflammatory cascade NF-κB and helps production of pro-inflammatory cytokines like TNF-α, IL- 6 [69, 70]. Here, we demonstrated that NF-κB and TNF- α levels were elevated significantly in scopolamine alone administered rats. While, pre-treatment with levosimendan significantly attenuated the upregulation of NF-κB and in response decreased the inflammatory mediators caused by the scopolamine administration showing its anti-inflammatory property (Fig. 10C, 10D). These results were in line with the previous studies stating that levosimendan ameliorated the scopolamine induced neuroinflammation by inhibiting NF-κB expression [71].
Cerebral vasculature is crucial for neuronal function as it is responsible for delivery of nutrients and removal of metabolites. Over production of amyloid beta plaques in the cerebral blood vessels worsens the disease pathology and contributes to cognitive decline [72]. According to the previous studies, muscarinic receptor blockage by scopolamine injection activates beta secretase activating cleavage enzyme – 1 (BACE-1). It cleaves amyloid precursor protein (APP) and causes Aβ over production [73]. In this study, the scopolamine alone administered group has shown a significant increase in Aβ deposition in the hippocampus which is similar to the previous studies [10, 25]. Whereas, our experiments in the present study proved the same trend that pretreatment with levosimendan effect on APP and BACE1 repression confirmed that its anti-AD effects are related to the inhibition of Aβ production. In addition, cilostazol does not affect LRP, which is a key Aβ clearance receptor and NEP and IDE involved in the degradation of the Aβ peptide [74, 75] (Fig. 10A, 11C). Previous studies demonstrated the neuroprotective effect of levosimendan i.e., Levijoki et al, 2015 reported that, levosimendan increases the cerebral blood volume and protects the brain against primary and secondary stroke [76]. Roehl et al, 2010 reported the neuroprotective effect of levosimendan in an in vitro model of traumatic brain injury [77]. These results are in line with previous studies supporting the beneficial effects of levosimendan as PDE inhibitor through an increase in Aβ aggregation by inhibiting BACE-1 and Aβ clearance.
The neurotrophin family, BDNF plays a pivotal role in the differentiation, survival, and functional maintenance of nerve cells. It is expressed all over the brain including astrocytes, glial cells and protects against disease causing pathologies by promoting neuronal survival. Aβ oligomers in the brain blocks the activation of CREB pathway and decreased the levels of BDNF. This reduced BDNF levels in the brain causes neurodegeneration that leads to many neurological diseases like AD [27]. In this study, scopolamine injection decreases the expression of BDNF that affects the neurogenesis and synaptic plasticity which is similar to previous studies [78]. Meanwhile, pre-treatment with levosimendan significantly increased the expression of BDNF. The possible protective action of levosimendan might be due to its inhibitory action on phosphodiesterase III enzyme. The inhibition of PDE III by levosimendan, increases the levels of cAMP. The cAMP binds to the regulatory subunit of protein kinase A (PKA) and causes the dissociation of PKA from the regulatory subunit. These dissociated PKA enters into the nucleus and phosphorylate the CREB. Further, the phosphorylated CREB bind with the cAMP responsive element in the promotor region of DNA and promotes BDNF expression [79, 80]. These results are consistent with previous studies that inhibition of PDE III enzyme stimulates pCREB/BDNF signalling and enhance neurogenesis [81].
Hence, based on these findings from behavioural and biochemical analysis, we demonstrate that levosimendan exerted its neuroprotective role against scopolamine induced cognitive dysfunction, oxidative stress, amyloid beta accumulation, BDNF decline and neuroinflammation.