Our research was the first to show that naproxen could have an additive neuroprotective effect in the AlCl3-induced Alzheimer's rat model when given simultaneously with rivastigmine, the standard anti-Alzheimer medication, by altering neuronal turnover, enhancing neurogenesis, and suppressing apoptosis. Naproxen has mitigated AlCl3-mediated neurocognitive deficit as evidenced by improved memory performance in the NOR test and reduced step-through latency in the PA test, besides ameliorating cholinergic deficits through decreased hippocampal AChE activity. Compared to rivastigmine alone, naproxen had no additional ameliorative influence on AD-related neurocognitive deficits when given synchronously with rivastigmine.
AD is an irreversible debilitating age-related neurologic disorder affecting over 47 million people worldwide. It is one of the most disappointing medical concerns that greatly distress societies and their economy (Colucci et al. 2014; Liu et al. 2014), especially as the population ages globally (Caselli et al. 2006; Arshavsky 2010). The disease hallmark is an insidious progressive intellectual decline that commonly involves the memory of recent facts, executive functions, and spatial orientation (Benedikz et al. 2009). Speech, mobility, and neuropsychiatric problems are among the patients' sufferings (Sun et al. 2013). Therefore, timely diagnosis plus achieving effective therapeutic approaches are mandatory.
AD is classically characterized by β-amyloid plaque deposits of Aβ peptides, activated microglia, reactive astrocytes, and NFTs (Beach et al. 1989; Iqbal and Grundke-Iqbal 2008; Weller and Budson 2018). Aβ brain aggregation, mediated through imbalance between their production and clearance, provokes subsequent pathological events as oxidative stress and neuroinflammation that eventually lead to neuronal loss (Mawuenyega et al. 2010; Mucke and Selkoe 2012; Liu et al. 2016). Activated microglia and reactive astrocytes, tightly associated with amyloid plaques (Olabarria et al. 2010), augment the local inflammatory response (Nagele et al. 2003; Rodríguez et al. 2009).
Memory and cognitive decline are directly correlated with cerebral cholinergic neuron degeneration within the basal forebrain (Whitehouse et al. 1981, 1982). Decreased acetylcholine (ACh) release, impaired choline acetyltransferase action, or increased activity of AChE further aggravate Ach scarceness in AD (Fishman et al. 1986; Hammond and Brimijoin 1988; Rodríguez-Puertas et al. 1994). NMDA (N-methyl-D-aspartate) receptors overactivation is fundamental for AD progression (Dingledine et al. 1999). ChEIs as tacrine, donepezil, rivastigmine, and galantamine (Ray and Lahiri 2009; Nazem et al. 2015) for mild to moderate circumstances and NMDA antagonists as memantine for moderate to severe cases are the currently approved therapy. They can only provide symptomatic relief, despite being partly effective in the early stages (Citron 2010; Alteri and Guizzaro 2018). The standard anti-Alzheime drug, rivastigmine (Birks and Evans 2015; Kandiah et al. 2017), can enhance intellectual functioning through raising synaptic ACh levels mainly within the hippocampus and neocortex (Gothwal et al. 2019).
A potential association between AD incidence and aluminum (Al) content in drinking water has been indicated (McLachlan et al. 1996; Altmann et al. 1999; Gauthier et al. 2000; Peder Flaten 2001; Exley and Esiri 2006). An elevated concentration of Al was detected in AD patients' brains (Crapper et al. 1973). Al industry personnel serving in miners, foundry, and welders have displayed impaired cognitive functions (Rifat et al. 1990; Polizzi et al. 2002; Giorgianni et al. 2003). Excess Al intake induces amyloid-beta precursor protein (APP) overexpression, amyloid deposition (Castorina et al. 2010), apoptosis activation besides degenerative neuronal changes (Crapper et al. 1973; Zatta et al. 2003; Kawahara and Kato-Negishi 2011).
Divergent aspects of AD have been studied through numerous categories of animal models. Rats are considered the favored species (Benedikz et al. 2009) as they are brainy, quick learners besides their similarities in physiological processes (Benedikz et al. 2009) and pathological alterations to human beings (Herrera et al. 1999; Reid et al. 2001; Hörsten et al. 2003; Loeffler 2004). We, therefore, decided to study the AlCl3 induced Alzheimer rat model as it is the most relevant to the sporadic AD pathology (Evrard et al. 1998; Li et al. 2016). Chronic AlCl3 administration enhances Al CNS access through the blood-brain barrier (BBB) via special high-affinity transferrin receptors (Roskams and Connor 1990). Al accumulates in all rat brain areas (Abubakar et al. 2004; Sakamoto et al. 2004; Kaur and Gill 2006), although the cerebral cortex, the hippocampus, and the cerebellum are the structures most commonly implicated in the lesions initiated by Al intoxication (Slanina et al. 1984; Misawa and Shigeta 1992). Our study showed significant neuronal degeneration presented as marked shrunken dark basophilic neurons with infiltration of glial cells within the CA1 pyramidal layer in the Al group, consistent with previous studies (Junior et al. 2013; Said and Rabo 2017; Bazzari et al. 2019). A significant increase in the deeply stained neurons within the Purkinje cell layer associated with electron-dense cytoplasm, ill-defined nuclei, and organelles within the Purkinje and granular cell layers were displayed within Al group cerebellar tissues. Our findings are in line with other studies (Bhalla and Dhawan 2009; Bondy 2016) that revealed disorganization in the architecture of the Purkinje cell layer with a loss of Purkinje cells of cerebellar cortex in the AD rat model. RIVA group revealed the more or less normal histological structure of the hippocampus CA1 field and cerebellum Purkinje cell layer with a substantial reduction in the number of dark or deeply stained cells that is in line with other research (Mahdy et al. 2012), confirming the mitigating impact of rivastigmine on AlCl3-induced neurotoxicity.
AlCl3 AD rat model displayed a biphasic response of AChE activity; an increase in AChE activity in short-term administration followed by pronounced decay of the enzyme activity on long-term (Kumar 1998; Kaizer et al. 2008). Long-term response ensues by a slow accumulation of Al with the formation of a complex with the anionic site of the AChE enzyme (Kumar 1998; Kaizer et al. 2008; Nampoothiri et al. 2015). In agreement with other studies (Zheng et al. 2009; Said and Rabo 2017), our findings revealed a significant increase in hippocampal AChE activity in the Al group indicating short phase response. Following previous research (Onor et al. 2007; Nampoothiri et al. 2015, 2017), our study showed that rivastigmine inhibited AlCl3-enhanced hippocampal AChE activity, which partially enhanced cholinergic neurotransmission. In advanced cases, however, rivastigmine exerts its action by inhibiting BuChE activity (Greig et al. 2001; Giacobini et al. 2002).
AlCl3-treated animals have demonstrated major memory and cognitive deficits (Ikram et al. 2020; Mustafa 2020). They showed increased retention latency and decreased RI percentage (Jangra et al. 2015), indicating decreased short, intermediate, and long-term memory. Intracerebral administration of AlCl3 triggered learning deficits in rabbits as well (Rabe et al. 1982). The NOR test revealed that the Al group had a week exploratory preference compared to the Cont group and degradation in spatial and retention memory as measured by PA test, which is consistent with previous literature (Lakshmi et al. 2015).
Rivastigmine attenuated AlCl3-induced spatial and retention memory deficit (Mehrabadi et al. 2020). Decreased escape latency and increased discrimination index were reported in the streptozotocin rat model of AD treated with rivastigmine (Akhtar et al. 2020). Moreover, rivastigmine exerted positive effects on learning and memory in the chronic d-galactose-induced accelerated aging rodent model (Chogtu et al. 2018). It reversed spatial learning deficits induced by scopolamine as well (Deiana et al. 2009).
Neuroinflammation is acknowledged as a prominent feature in AD pathology (Hensley 2010). Aβ directly activates glial cells, astrocytes and microglia, found near Aβ plaques (Perlmutter et al. 1992; Townsend and Praticò 2005; Schwab and McGeer 2008; Heneka et al. 2015), triggering the release of inflammatory mediators, notably IFNγ, IL-1β, TNF-α, IL-6, TGF-β (Constam et al. 1992; McGeer and McGeer 1995; Hu et al. 1998; Johnstone et al. 1999), free radicals, inducible nitric oxide synthase (iNOS) and nitric oxide (NO) (Hu et al. 1998; Ayasolla et al. 2004; Garção et al. 2006; Furman et al. 2012; Cai et al. 2014). It is believed that reactive astrogliosis, hypertrophy of cell soma, and processes induced by Aβ plays a role in limiting the build-up of Aβ plaques (Olabarria et al. 2010; Hou et al. 2011) through the expression of type III intermediate filament (IF) protein; glial fibrillary acidic protein (GFAP) (Wilhelmsson et al. 2006; Kamphuis et al. 2012). On the contrary, astrogliosis could enhance neurotoxicity with further neuronal death (Scuderi et al. 2014). Its role, therefore, in AD is still controversial. Nevertheless, astrogliosis degree is usually correlated with cognitive decline in AD (Beach and McGeer 1988; Mrak et al. 1996; Kashon et al. 2004).
Our study detected an apparent increase in hippocampal GFAP immunoreactivity in AlCl3-intoxicated rats suggesting severe astrocytic activation induced by Al uptake. Our results are consistent with those of other studies (Guo-ross et al. 1999; Li et al. 2009; Erazi et al. 2010; Justin-Thenmozhi et al. 2018), which detected increased GFAP immunolabelling in the hippocampus and frontal cortex after chronic Al intoxication in rats and rabbits (Yokel and Callaghan 1998) though unchanged GFAP immunoreactivity was still reported (Platt et al. 2001). Other studies, on the contrary, revealed a decrease in GFAP immunoreactivity, reflecting astrocytes' susceptibility to Al-induced neurotoxicity (Guo-ross et al. 1999; Junior et al. 2013). RIVA group showed enhanced GFAP immunoreactivity indicating persistent astrogliosis, which contradicts other studies (Mohamed et al. 2016) that displayed reduced immunoreactivity by 45–50%. Similarly, rivastigmine has attenuated the memory decline in T2DM-AD model mice by suppressing gliosis (Matsuda and Hisatsune 2017). The diversity of the AD models may lie behind the conflicting outcomes.
Microglia were studied to play dual roles in Aβ pathogenesis (Ard et al. 1996; Grathwohl et al. 2009; Majumdar et al. 2011). Early microglial recruitment enhances Aβ clearance across the BBB via microglia scavenger receptors (Khoury et al. 1996; Paresce et al. 1996; Kunjathoor et al. 2004; Alarcón et al. 2005; Yang et al. 2011). Chronic inflammation, however, mediates microglial dysfunction, which plays a detrimental effect via the release of cytotoxic molecules, enhanced production, and impaired clearance of Aβ (Hickman et al. 2008; Brandenburg et al. 2010; Feng et al. 2011). This is likely mediated by decreased expression of enzymes that degrade Aβ; insulysin, neprilysin, and metallopeptidase 9 matrix (MMP9); and loss of functional and structural integrity BBB (Hickman et al. 2008; Brandenburg et al. 2010; Feng et al. 2011).
A vicious cycle of inflammation has been collectively designed between Aβ accumulation, activated microglia, and microglial inflammatory mediators. Therefore, a therapeutic intervention targeting neuroinflammation, particularly in early disease processes (McGeer et al. 1996; Stewart et al. 1997; Vlad et al. 2008), seems to potentially restrain AD's further progression. NSAID use is associated with a lower risk of developing AD in a range of ethnodemographic populations (Breitner et al. 1995; Stewart et al. 1997; Veld et al. 2001; Landi et al. 2003; Fischer et al. 2008; Szekely et al. 2008; Vlad et al. 2008; Côté et al. 2012) besides various preclinical studies (Lim et al. 2000; Weggen et al. 2001; Yan et al. 2003). Importantly, a greatly reduced risk of AD was noticed in rheumatoid arthritis patients on long-term NSAIDs (Mcgeer et al. 1990). Moreover, chronic therapy improved behavioral deficits in AD rodent models (McGeer and McGeer 2007). NSAIDs acts through inhibiting cyclooxygenase (COX) (Varvel et al. 2009), in particular, COX-1, as it is primarily expressed in microglia (Deardorff and Grossberg 2017), leading to blockade of microglial activation besides altered immune cells infiltration. COX-1 deficient mice exhibit reduced inflammation and neuronal injury levels in response to Aβ (Choi and Bosetti 2009). NSAIDs can act as α-, β-, and γ-secretase modulators, which help lessen amyloid deposition (Kukar and Golde 2008). Furthermore, they can directly interact with the Aβ peptide inhibiting the formation of Aβ oligomers and deposits (Kukar et al. 2008), which consequently attenuate microglial activation, the number of reactive astrocytes, and expression of proinflammatory molecules (Lim et al. 2000; Jantzen et al. 2002; Richardson et al. 2002; Yan et al. 2003; Heneka et al. 2005; Kotilinek et al. 2008). PPAR-gamma agonists as ibuprofen, indomethacin, and naproxen decrease promoter activity of the beta-site APP-cleaving enzyme 1 (BACE1) involved in APP processing as well (Sastre et al. 2006). Therefore, NSAIDs' beneficial effects have been attributed to their ability to reduce Aβ generation, plaque size, and tau phosphorylation (Yoshiyama et al. 2007; Khoury and Luster 2008) with subsequent improvement in behavioral impairments (Lim et al. 2000, 2001; Sung et al. 2004; Kukar et al. 2007).
NSAIDs can prevent the intellectual decline in older adults if started earlier, before age 65 (Hayden et al. 2007). Existing cognitive impairments limit their therapeutic usefulness (Martin et al. 2008). Ibuprofen and naproxen can block the early appearance of neuronal ectopic cell cycle events (CCEs), an early marker for risk of neurodegeneration (Busser et al. 1998; Yang et al. 2001, 2003), however, failed to reverse the current events in older mice (Varvel et al. 2009). Unexpectedly, NSAIDs can accelerate AD pathogenesis in advanced cases (Breitner et al. 2009) possibly, through inhibiting microglia-mediated clearance of Aβ and the compensatory neurogenesis processes.
Naproxen, a common over-the-counter medicine, could help to prevent the progression of AD (Kim et al. 2011) through destabilizing preformed Aβ fibrils, reducing their amounts (Agdeppa et al. 2003; Hirohata et al. 2005) besides antagonizing Aβ aggregation (Cole and Frautschy 2010). Naproxen chronic administration blocked alterations in brain microglia and neuronal cell cycle events in young transgenic animals (Imbimbo et al. 2010). Long-term prophylactic use may reduce the risk of AD by 67% compared to placebo (Vlad et al. 2008; Imbimbo 2009; Imbimbo et al. 2010), though; it offers no therapeutic influence in preexisting AD cases (Gasparini et al. 2004; Imbimbo 2004). Our findings established the ability of naproxen to restore the architecture of pyramidal and Purkinje cells, although some of them were still shrunk, irregular, and deeply stained. Compared to the RIVA group, the RIVA+Napro proved to have an additive neuroprotective effect on the hippocampal pyramidal cells but could not provide extra benefit to the cerebellar Purkinje cells.
In our research, naproxen had a mitigating effect over AlCl3 anticholinergic action through decreased AChE activity. Hence, our findings agree with previous studies (Mostafa et al. 2016) that showed naproxen's ameliorative effect on AD-like behavioral performance through attenuating AChE activity. Our findings confirmed naproxen's ability to alleviate intellectual dysfunction caused by AlCl3 as evidenced by significantly augmenting the time exploring the novel objects, step-through latency, and spatial learning compatible with other studies' results (Jain et al. 2002). Chronic treatment with naproxen significantly improved colchicine-induced cognitive impairment (Kumar et al. 2006). Moreover, naproxen has potential neuroprotective properties against D-serine mediated excitotoxicity implicated in PD, Huntington's disease, multiple sclerosis, and AD (Armagan et al. 2012). Some reports, however, have argued against naproxen anti-Alzheimer role (Lyketsos et al. 2007; Martin et al. 2008; Imbimbo et al. 2010). When compared to the RIVA group, the RIVA+Napro group was unable to provide an additive cognitive improvement.
AD neuronal cell death can be attributed to apoptosis and DNA fragmentation (Chang et al. 2016). Disruption of mitochondrial homeostasis by high levels of reactive oxygen species (ROS) mediates the release of proapoptotic cytokines as cytochrome c (Tyagi et al. 2006; Liu et al. 2015) that bind to apoptotic protease activating factor 1 (Apaf-1) (Skulachev 1998; Ghribi et al. 2002; Al-olayan et al. 2015; Resseguie et al. 2015). This complex activates caspase-3, the major executioner in apoptosis (Lynch et al. 2000; Sjöbeck and Englund 2001; Chang et al. 2016), via proteolytic cleavage (Allan and Clarke 2009; Reubold and Eschenburg 2012) into two subunits which dimerize to form the active enzyme (Rotonda et al. 1996). Aβ can induce neuronal apoptosis via activation of caspase-3 found colocalized within senile plaques of AD brains (Amelio et al. 2012; Cetin et al. 2013; Chang et al. 2016). Besides, studies have illustrated an apoptogenic role of AChE (Zhang and Greenberg 2012), which revealed the potential value of AChEIs therapeutics in early AD (Toiber et al. 2008).
Our study showed that the protein level of the activated caspase‐3 was significantly augmented in AlCl3‐intoxicated rats in the hippocampus and cerebellar tissues compared with the Cont group, which is in line with other research (Greilberger et al. 2008; Porsteinsson et al. 2008; Kumar and Kumar 2009; Al-olayan et al. 2015; Alawdi et al. 2017; Justin-Thenmozhi et al. 2018; Attia et al. 2020). AlCl3-induced activated caspase‐3 overexpression was significantly attenuated by the concomitant treatment with rivastigmine. This is in line with other study reports that showed an ameliorating effect of rivastigmine over AD-mediated caspase-3 overexpression (Elmegeed et al. 2015; Sachdeva and Chopra 2015). Similarly, activated caspase-3 overexpression caused by AlCl3 was significantly attenuated by concomitant naproxen administration. NSAIDs are competitive multi-caspase inhibitors, but their actions are more pronounced for caspases-4, -5, and -9, with lower activity against caspase-3 and -1 due to variations in the pocket recognition substrates (Smith et al. 2017). Caspase inhibition is recognized as a Cox-independent (Chan 2002) anti-inflammatory mechanism for NSAID drugs with a consequent decrease in cell death and proinflammatory cytokine production (Smith et al. 2017). When naproxen was given with rivastigmine, it had an additive antiapoptotic effect on the hippocampal CA1 region compared to rivastigmine alone. Still, according to our findings, it failed to have an additive antiapoptotic to the cerebellar Purkinje cells. On the contrary, other studies showed the ability of NSAIDs to induce apoptosis which lies behind their chemopreventive impact against cancer of the gastrointestinal tract (gastric or colorectal cancer) (Chan 2002; Jana 2008) lung, breast, and prostate cancers (Chan et al. 2005; Rothwell et al. 2010, 2012; Shebl et al. 2014; Seetha et al. 2020).
Recent studies have suggested the implication of neurogenesis in neurodegenerative disorders (Zhao et al. 2008; Lazarov et al. 2010; Mu and Gage 2011; Marxreiter et al. 2013). Deficits in adult neurogenesis may contribute to tau hyperphosphorylation in new neurons, compromised hippocampal circuitry, and cognitive impairments in AD (Hollands et al. 2017). Therefore, through pharmacological and genetic approaches, induction of neurogenesis can slow down disease progression (Cho et al. 2007). The neurogenesis process has been well acknowledged in two brain regions; the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus DG (Zhao et al. 2008). Nevertheless, neurogenesis has still been specified in other regions of the adult mammalian brain as the neocortex, cerebellum, striatum, amygdala, and hypothalamus (Radad et al. 2017). The cerebellum encloses much of the adult brain's mature neurons (Wingate and Hatten 1999). It has a remarkable feature of being able to regenerate its cells by neurogenesis following damage (Andreotti et al. 2018). Strikingly, after its ablation by irradiation, the cerebellar external granular layer can be reconstituted (Altman et al. 1969) probably through cerebellar nestin-expressing progenitors, residing mostly in the Purkinje cell layer, that can differentiate into granule cell precursors (GCPs) and mature granule neurons (Wojcinski et al. 2017). Our study showed a significant reduction of nestin immunoreactivity in the granule cell layer of the Al group compared to the Cont group indicating impaired cellular proliferation that is consistent with other studies (Yu et al. 2018; Li et al. 2019; Ibrahim et al. 2021). However, concomitant administration of rivastigmine increased nestin immunoreactivity, presumably, due to compensation for cholinergic deficits (Tayebati et al. 2004). This was consistent with other research findings (Salem et al. 2014) that showed enhanced expression of the brain nestin gene by 65.2% through rivastigmine administration relative to the untreated AD population. Similarly, naproxen administration-induced nestin protein overexpression may represent a therapeutic option to restore adult neurogenesis in AD patients. Previous studies showed the potential effectiveness of one of the non-selective COX inhibitors, indomethacin, to restore adult neurogenesis in PD (Hain et al. 2018). When concurrently given with rivastigmine, naproxen exerted an additive effect in promoting neurogenesis relative to rivastigmine-only therapy. Therefore, it could possibly enhance rivastigmine anti-Alzheimer's activity.