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. 2023 Jan 3;8(2):2227–2236. doi: 10.1021/acsomega.2c06467

Magnoflorine-Loaded Chitosan Collagen Nanocapsules Ameliorate Cognitive Deficit in Scopolamine-Induced Alzheimer’s Disease-like Conditions in a Rat Model by Downregulating IL-1β, IL-6, TNF-α, and Oxidative Stress and Upregulating Brain-Derived Neurotrophic Factor and DCX Expressions

Dar Junaid Bashir †, Saliha Manzoor †, Mohd Sarfaraj †, Shekh Mohammad Afzal ‡, Masarat Bashir §, Nidhi ∥, Shweta Rastogi ⊥, Indu Arora #, Mohammed Samim †,*
PMCID: PMC9850486  PMID: 36687096

Abstract

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Dementia or the loss of cognitive functioning is one of the major health issues in elderly people. Alzheimer’s disease (AD) is one of the common forms of dementia. Treatment chiefly involves the use of acetylcholinesterase (AChE) inhibitors in AD. However, oxidative stress has also been found to be involved in the proliferation of the disease. Magnoflorine is one of the active compounds of Coptidis Rhizoma and has high anti-oxidative properties. Active principle-loaded nanoparticles have shown increased efficiency for neurodegenerative diseases due to their ability to cross the blood–brain barrier more easily. An in vitro study involving magnoflorine-loaded chitosan collagen nanocapsules (MF-CCNc) has shown them to possess inhibitory effects against oxidative stress and to some extent on AChE as well. In the current study, both nootropic and anti-amnesic effects of magnoflorine and MF-CCNc on scopolamine-induced amnesia in rats were evaluated. The treatment was done intraperitoneally (i.p.) once daily for 17 consecutive days with MF-CCNc (0.25, 0.5, and 1 mg), magnoflorine (1 mg), and donepezil (1 mg). To induce amnesia, hence, cognitive deficit rats were induced with scopolamine (1 mg/kg) daily for the last 9 days. Novel object recognition (NOR) and elevated plus maze (EPM) behavioral analysis were done to assess memory functioning. Hippocampal tissues were extracted to study the effect on biochemicals (AChE, MDA, SOD, and CAT), pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), and immunohistochemistry (brain-derived neurotrophic factor (BDNF) and DCX). MF-CCNc showed memory-enhancing effects in nootropic as well as chronic scopolamine-treated rats in NOR and an increase in inflexion ratio in EPM. MF-CCNc reduced the levels of AChE and MDA while increasing SOD and CAT levels in the hippocampus. MF-CCNc further lowered the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. These nanocapsules further increased the expression of BDNF and DCX that are necessary for adult neurogenesis. From the research findings, it can be concluded that MF-CCNc has high anti-amnesic properties and could be a promising candidate for the treatment of AD.

1. Introduction

Alzheimer’s disease (AD) is an age-related cognitive disorder and is linked to the large decrease in memory and learning functions, as a result of loss of neuronal functions in the individuals suffering from it.1−3 AD is one of the most common forms of dementia and is responsible for cognitive dysfunction in elderly people worldwide.4,5 AD arises in mid to late adult life and is characterized by defects in memory and cognitive functions like reasoning and performing complex learned motor tasks (apraxias). Behavioral disturbances like agressivity, disinhibition, and speech disturbances overshadow clinical syndrome in the fronto-temporal dementias arising from the involvement of the frontal neocortex. The gradual amassing of the beta-amyloid protein fragment (senile plaques) and hyperphosphorylated, aggregated tau proteins (neurofibrillary tangles) is the key features of AD pathogenesis.6 Beta-amyloid plaques intervene in neuron to neuron communication at synapses and act as a neurotoxin. On the other hand, tau is responsible for intracellular trafficking and prevents the moment of essential molecules and nutrients upon hyperphosphorylation.7,8 A classical neuropathological sign of AD is the extracellular agglomeration of Aβ peptides as senile plaques. The sequential cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase leads to a diseased form of AD.9 Studies have largely focused on APP metabolism, Aβ toxicity, and tau phosphorylation. The focus has now shifted to other pathological features for better understanding; these include mitochondrial dysfunction, oxidative stress, insulin resistance, and inflammation pathways.10 Neuroimaging initiatives and other longitudinal studies suggest that Aβ deposition alone does not lead to AD, and it is rather a multifactorial disease.11,12 “Mitochondrial cascade hypothesis” suggests that the more prevalent, sporadic late-onset AD is due to the primary insult caused by mitochondrial dysfunction.13,14

Natural products are profoundly used as therapeutics for neurodegenerative diseases. The structural diversity of plant alkaloids makes them suitable as therapeutic agents. Like other neurodegenerative diseases, alkaloids are being used in AD. The two alkaloids that are currently used as a treatment for AD are galantamine and rivastigmine. Other alkaloids that have shown efficacy for AD are huperzine A, berberine, caffeine, indomethacin, and indirubin.15 The current study involved the use of magnoflorine, an aporphine alkaloid that possesses antioxidant and antiradical properties.16 The passive avoidance test of magnoflorine has shown that it possesses cognition and antiamnestic properties.17 Antioxidant and antiradical properties of magnoflorine together with cognitive properties make it a suitable candidate for AD.

The blood–brain barrier (BBB) is primarily a diffusion barrier; the influx of most compounds from the blood to the brain is carried out through this barrier in the brain.18 Therapeutics for AD and other related neurodegenerative diseases are limited by this barrier. To cross the BBB, invasive ways like ultrasound-based delivery, intracerebral or intracerberoventricular infusion/injection, and non-invasive approaches like the pro-drug strategy and nanocarrier strategy are being used. The nanocarrier strategy that involves the use of nanoparticles, dendrimers, liposomes, and solid-lipid nanoparticles has found great applicability in drug delivery for neurodegenerative diseases, partly because they do not lead to any major disruption in the integrity of the BBB.19 The ability of biodegradable polymer-coated active principle-loaded nanoparticles like those of chitosan and Tween 80 to cross the BBB easily in comparison to the active principles makes them more suitable for research on neurodegenerative diseases.20−22

Our previous work involved the in vitro studies of magnoflorine (MF) and magnoflorine-loaded chitosan collagen nanocapsules (MF-CCNc) and showed that both MF and MF-CCNc have inhibitory effects on oxidative stress.23 In the present work, we examined the anti-amnesic potential of MF and MF-CCNc on memory deficits caused by the scopolamine-induced rat model (Scheme 1).

Scheme 1. Pathways of Action of MF-CCNc on Alzheimer-like Scopolamine-Induced Amnesia.

Scheme 1

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2. Results and Discussion

2.1. Behavioral Study

Nootropic and scopolamine-induced anti-amnesic behavioral studies were done using novel object recognition (NOR) and elevated plus maze (EPM) behavioral tests. The NOR test allows assessment of visual recognition memory related to AD and is affected during the early progression of the disease.24 For the assessment of long-term spatial memory, EPM was used.25

2.1.1. Nootropic Effect of MF and MF-CCNc

Figure 1a,c shows the results obtained from the nootropic study of MF, and the MF-CCNc NOR test was done after pre-treating rats with MF and different doses of MF-CCNc for 7 days to assess the effect on memory function. The results so obtained were expressed in the form of the percentage recognition index. Percentage recognition of MF and MF-CCNc was found to be higher than both control and donepezil (Dpz) groups, depicting the nootropic behavior of MF and MF-CCNc. It was observed that MF showed increased nootropic activity with a p-value of p < 0.001. Dpz showed non-significant (ns) nootropic activity in comparison to control, while MF-CCNc (0.25 mg), MF-CCNc (0.5 mg), and MF-CCNc (1 mg) were statistically significant with p-values of p < 0.001, p < 0.05, and p < 0.0001, respectively. It was further observed that at 1 mg/kg concn, MF-CCNc has a higher recognition index in comparison to both Dpz and MF. Although the p-values were significant (p < 0.01) for Dpz, however, it was ns in comparison to MF. EPM was expressed in the form of an inflexion ratio (Figure 1c), and an increase in the values of the inflexion ratio was observed for both MF and MF-CCNc in comparison to control and Dpz. Results of MF-CCNc (1 mg) showed that there is a significant increase in the inflexion ratio in comparison to Dpz with a p-value of p < 0.05; however, it was non-significant in comparison to MF. It can be concluded from the nootropic study that MF and MF-CCNc can amplify memory function in rats.

Figure 1.

Figure 1

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Novel object recognition (NOR) and elevated plus maze (EPM) behavioral analysis of treated animals in nootropic and scopolamine models. (a) Plot of NOR for the nootropic model represented in the form of the recognition index. (b) Plot of NOR for the scopolamine model represented in the form of the recognition index. (c) Plot of NOR for the nootropic model represented in the form of the inflexion ratio. (d) Plot of NOR for the scopolamine model represented in the form of the inflexion ratio. Data obtained is expressed as mean ± SEM (n = 6), which is followed by statistical analysis with one-way ANOVA and Tukey test; based on p-values, ns is used when p > 0.05 with respect to control and negative control (SCP); similarly, other values of p are represented as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical analysis of MF-CCNc (1 mg) with respect to MF (1 mg) and Dpz (1 mg), ns p > 0.05, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001.

2.1.2. Anti-Amnesic Effect of MF and MF-CCNc in Rats with Scopolamine-Induced Amnesia

The scopolamine-induced dementia model has been used for AD recently to assess the potential of therapeutic agents.26 Scopolamine was induced into the rats after completion of the nootropic study, and NOR and EPM tests were done. Scopolamine is a non-selective muscarinic cholinergic receptor antagonist and causes cholinergic dysfunction and hence learning and memory deficits. Scopolamine has shown previously that it can lead to spatial memory deficit by disrupting cholinergic neurotransmission.27,28 Similar results were found in our study; scopolamine decreased the inflexion ratio and recognition index, thereby affecting special memory and cognitive deficit. Donepezil that is used to treat patients suffering from AD was used as a positive control. Donepezil has shown the ability to reverse scopolamine-induced memory impairment in previous studies.29,30 In the present study, a decrease in the recognition index was observed in the NOR test of the scopolamine group (negative control) in the chronic scopolamine amnesia model, as shown in Figure 1b. MF and all three groups of MF-CCNc showed a higher recognition index. A statistically significant difference in the recognition index was observed in all the groups in comparison to a negative control group with a p-value of (p < 0.0001). At a similar concn of 1 mg/kg body weight of animals, MF-CCNc showed a significant increase in the recognition index in comparison to Dpz and MF with p-values of p < 0.001and p < 0.05, respectively. Similarly, all the groups showed a statistically significant increase in the inflexion ratio, depicting an increase in the retention memory (p < 0.0001). MF-CCNc showed a significant increase in the inflexion ratio in comparison to Dpz and MF with p-values corresponding to p < 0.0001. These results depict that the MF-loaded chitosan collagen nanocapsules increase the spatial and retention memory significantly in rats induced with scopolamine.

2.2. Effect of MF and MF-CCNc on Hippocampal AChE in Rats

Acetylcholine is one of the vital neurotransmitters involved in the maintenance of cognitive functions. Dementia is characterized by the loss of cholinergic neurons in parts of the brain.31 Acetylcholine is degraded by AChE, and overproduction of it leads to reduction in acetylcholine and memory impairment thereof. This also leads to a substantial increase in the activity of AChE. An increase in the activity of AChE in the brain of scopolamine-induced rats has been reported.32,33 In the present study, also, the activity of AChE increased significantly in the scopolamine group in comparison to the SCP group with a p-value of (p < 0.0001). Results further showed that MF-CCNc (1 mg) reduced the activity of AChE in comparison to MF, and the lowering in activity was significant in comparison to MF with a p-value of (p < 0.001). The results were evaluated using one-way ANOVA followed by the Tukey test, as depicted in Figure 2a.

Figure 2.

Figure 2

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Effect of MF and MF-CCNc on biochemical parameters: (a) acetylcholinesterase (AChE), (b) malondialdehyde (MDA), (c) superoxide dismutase (SOD), and (d) catalase (CAT) in scopolamine-induced rats. Data obtained is expressed as mean ± SEM (n = 3), which is followed by statistical analysis with one-way ANOVA and Tukey test based on p-values; ns is used when p > 0.05 with respect to control and negative control (SCP); similarly, other values of p are represented as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistical analysis of MF-CCNc (1 mg) with respect to MF (1 mg) and Dpz (1 mg), ns p > 0.05, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001.

2.3. Effect of MF and MF-CCNc on MDA, SOD, and Catalase Levels

Oxidative stress that is developed by reactive oxygen species (ROS), reactive nitrogen species (RNS), and free radicals is believed to play a vital role during the progression of AD. Scopolamine is believed to increase oxidative stress by increasing the levels of MDA and lowering the levels of oxidative parameters.34,35 MF, Dpz, and all three groups of MF-CCNc reduced the levels of MDA significantly with a p-value of (p < 0.0001) when compared to the negative control, i.e., the SCP group. MF-CCNc showed a significant reduction in the levels of MDA in comparison to MF and Dpz with a p-value corresponding to (p < 0.0001), as depicted in Figure 2b. Further, the levels of SOD and CAT were lower in the SCP group. However, the levels of SOD and CAT increased significantly upon treatment of scopolamine-induced rats with Dpz, MF, and MF-CCNc with a p-value of (p < 0.01), (P < 0.0001) as depicted in Figure 2c,d. A significant increase in the levels of SOD and CAT was found in MF-CCNc in comparison to Dpz and MF. The p-values were found to be P < 0.0001 in SOD and (p < 0.01) and (p < 0.05) in catalase corresponding to Dpz and MF, respectively.

2.4. Effect of MF and MF-CCNc on Hippocampal-Based Pro-Inflammatory Cytokines in Rats

The release of neuroinflammatory cytokines IL-1β, IL-6, and TNF-α is one of the prominent features observed during the development of AD.36 A significant increase in the levels of IL-1β, IL-6, and TNF-α was observed in the SCP group, whereas Dpz, MF, MF-CCNc (0.25 mg), MF-CCNc (0.5 mg), and MF-CCNc (1 mg) depicted a significant decrease in the levels of these cytokines with a p-value of (p < 0.0001). It was further observed that MF-CCNc decreased the levels of these cytokines significantly in comparison to standard drug Dpz and MF. The p-value of MF-CCNc in comparison to MF and Dpz in IL-1β is (p < 0.0001), as shown in Figure 3. A significant decrease in the level of IL-6 of MF-CCNc in comparison to MF was observed with a p-value of (p < 0.05); however, the decrease was non-significant in comparison to Dpz. The level of TNF-α was also reduced significantly in comparison to MF and Dpz with a p-value corresponding to (p < 0.01). These results suggest that MF-CCNc can act as a neuroprotective agent.

Figure 3.

Figure 3

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Effect of MF and MF-CCNc on cytokine levels: (a) IL-1β, (b) IL-6, and (c) TNF-α in scopolamine-induced rats. Data obtained is expressed as mean ± SEM (n = 6), which is followed by statistical analysis with one-way ANOVA and Tukey test, with respect to negative control (SCP); the p-value is denoted as ****P < 0.0001. Statistical analysis of MF-CCNc (1 mg) with respect to MF (1 mg) and Dpz (1 mg), ns p > 0.05, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001.

2.5. Immunohistochemistry Studies

2.5.1. Effect on BDNF Levels by MF and MF-CCNc

One of the most important regions in the brain critical for learning and memory function is the hippocampus. It is susceptible to molecules such as scopolamine that triggers spatial memory impairment by disrupting cholinergic neurotransmission.32 Certain neurotrophic factors modulate the adult hippocampal neurogenesis and neuroplasticity, and one such factor is BDNF.37 It was back in 1991 when it was evaluated that the reduced expression of BDNF may contribute to progressive cell death, one of the main characteristics of Alzheimer’s disease.38 Brain-derived neurotrophic factor (BDNF) enhances cholinergic neurotransmission. In patients suffering from AD, the expression of BDNF is lowered.39,40 Both MF and MF-CCNc showed a substantial increase in the expression of BDNF in comparison to negative control SCP. A significant increase in the level of BDNF was observed in the MF-CCNc (1 mg) group with a p-value of (p < 0.0001) (Figure 4a–h). Further, the results showed that in comparison to both MF and Dpz at a similar concn, MF-CCNc increased the expression of BDNF significantly.

Figure 4.

Figure 4

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Effects of MF, different concentrations of MF-CCNc, and DPZ on BDNF in the hippocampus (40×) (BDNF, immunostaining). (a) BDNF expression of control exhibited in the form of blue coloration in the hippocampal neurons; (b) BDNF expression of scopolamine; (c) BDNF expression of Dpz; (d) BDNF expression of magnoflorine; (e) BDNF expression of manoflorine-loaded chitosan collagen nanocapsules (MF-CCNc) (0.025 mg); (f) BDNF expression of MF-CCNc (0.5 mg); (g) BDNF expression of MF-CCNc (1 mg); (h) BDNF IHC histogram. Data obtained is expressed as mean ± SEM (n = 3), which is followed by statistical analysis with one-way ANOVA and Tukey test, with p-values of *P < 0.05, ***P < 0.001, and ****P < 0.0001. Statistical analysis of MF-CCNc (1 mg) with respect to MF (1 mg) and Dpz (1 mg), ns p > 0.05, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001.

2.5.2. Effect on DCX Levels by MF and MF-CCNc

Neurogenesis plays a vital role in the hippocampal memory function in adults.41 Production of new neurons in the hippocampus continuously is necessary for memory function.42 The production of these new neurons is done by adult neural stem cells (NSCs) that are found in the subgranular region of the dentate gyrus region of the hippocampus.43 DCX is a marker of neuronal cells in the adult hippocampus. It is used as a marker of newly formed neurons in the dentate gyrus region (DG). DCX is continuously expressed during adult neurogenesis as it is involved in the development and migration of neurons.44,45 Its expression is believed to be decreased during aging, resulting in a decrease in neurogenesis.46 DCX staining of the scopolamine-induced rat hippocampus treated with MF, DPZ, and different concentrations of MF-CCNc was done in the present work. Significantly reduced levels of immature neurons were found in the SCP group. Treatment with MF and its nanocapsules (MF-CCNc) ameliorates repression of neuronal precursor cells of the subgranular region with the p-value corresponding to (p < 0.0001), as depicted in Figure 5a–h. MF-CCNc showed a significant increase in the expression of DCX in comparison to both MF and Dpz.

Figure 5.

Figure 5

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DCX immunohistochemical analysis of the effects of MF and MF-CCNc in reducing the suppression of neurogenesis in the dentate gyrus region of the hippocampal tissue of scopolamine-induced rats (40×) (DCX, immunostaining). (a) DCX expression of control exhibited in the form of blue coloration in hippocampal neurons; (b) DCX expression of scopolamine; (c) DCX expression of donepezil in scopolamine-induced rats; (d) DCX expression of magnoflorine in scopolamine-induced rats; (e) DCX expression of magnoflorine-loaded chitosan collagen nanocomposites (MF-CCNc) (0.25 mg); (f) DCX expression of MF-CCNc (0.5 mg); (g) DCX expression of MF-CCNc (1 mg); (h) DCX IHC histogram. Data obtained is expressed as mean ± SEM (n = 3), which is followed by statistical analysis with one-way ANOVA and Tukey test, with p-values of **P < 0.01 and ****P < 0.0001. Statistical analysis of MF-CCNc (1 mg) with respect to MF (1 mg) and Dpz (1 mg), ns p > 0.05, #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001.

3. Conclusions

It can be concluded from the above study that both magnoflorine and magnoflorine-loaded chitosan collagen nanocapsules (MF-CCNc) possess nootropic and neuroprotective properties in scopolamine-induced amnesia in rats, and MF-CCNc significantly ameliorates these properties. Nootropic effects can be attributed to the improvement of memory functioning due to the increase in recognition index and inflexion ratio on pretreatment with magnoflorine and magnoflorine-loaded chitosan collagen nanocapsules. It was further evaluated from the results that MF-CCNc possesses anti-amnesic properties. MF-CCNc significantly decreased the activity of acetylcholinesterase, responsible for the reduction of acetylcholine, which is one of the important factors for neurotransmission. MF-CCNc showed a significant decrease in the levels of MDA and an increase in the levels of SOD and CAT that are responsible for oxidative stress. MF-CCNc further showed an improvement in the adult hippocampal neurogenesis. Therefore, it can be concluded that magnoflorine-loaded chitosan collagen nanocapsules can be a great prospect for the treatment of diseases like Alzheimer’s and other forms of dementia.

4. Materials and Methods

Chemicals procured from Sigma-Aldrich were chitosan, malondialdehyde (MDA), dimethyl-sulfoxide (DMSO), and magnoflorine, the chemical procured from Spectrochem was 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide-hydrochloride (EDC·HCL), the chemical procured from SD Fine was Tween 80, and chemicals procured from Himedia Laboratories were 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB) and acetylthiocholine iodide (ATCl). Kits procured from Abbkine were IL-1β, IL-6, and TNF-α.

4.1. Animal Care

For the animal studies, 6–8 week old male Wistar rats (weighing 150–200 g) were used. They were divided into groups of six per cage and housed in the animal house facility of Jamia Hamdard, New Delhi. They were habituated under constant temperature (23 ± 1 °C), humidity (55 ± 3 °C), and 12:12 h light/dark cycle and were provided free access to water ad libitum. The acclimatization of the animals was allowed 1 week prior to experimentation to reduce stress. For all the experimental work, the procedures approved by the Animal Ethics Committee of Jamia Hamdard, New Delhi, India were used.

4.2. Experimental Design

MF-CCNc nanocapsules were prepared according to our previous method.23 Magnoflorine and MF-CCNc were solubilized in DMSO followed by saline. Both negative controls, i.e., scopolamine and donepezil, were prepared in saline. The control rats were administered normal saline throughout the experimentation. Treatment was given intraperitoneally (i.p.) at a volume corresponding to 0.1 mL/100 g of body weight for all the animals.27

A total of 42 animals were used for the experimentation, and they were divided into seven groups with six animals in each group. For nootropic and scopolamine models, a behavioral study was done.

4.3. Nootropic Model

Group 1: control (saline), group 2: positive control (donepezil (DPZ) 1 mg/kg), group 3: low dose of MF-CCNc 0.25 mg/kg, group 4: medium dose of MF-CCNc 0.5 mg/kg, group 5: high dose of EB-CCNc 1 mg/kg, and group 6: magnoflorine (MF) 1 mg/kg with n = 6 for all the groups.

To evaluate the nootropic activity of the animals in different groups, pretreatment of the above mentioned active principle and its nanocomposites in addition to saline and DPZ in the other two groups was given via the intraperitoneal route for eight consecutive days. The animals were subjected to behavioral tests NOR and EPM from day 6 until day 8.

4.4. Scopolamine Model

Group 1: control (saline), group 2: positive control (donepezil (DPZ) 1 mg/kg, scopolamine (SCP) 1 mg/kg), group 3: low dose of MF-CCNc 0.25 mg/kg (SCP 1 mg/kg), group 4: medium dose of EB-CCNc 0.5 mg/kg (SCP 1 mg/kg), group 5: high dose of EB-CCNc 1 mg/kg (SCP 1 mg/kg), group 6: epiberberine (EB) 1 mg/kg (SCP 1 mg/kg), and group 7: negative control (scopolamine (SCP) 1 mg/kg) with n = 6 for each group.

For the evaluation of scopolamine activity, scopolamine (1 mg/kg) was administered intraperitoneally (i.p.) after the pretreatment of the active principle and its nanocomposites, and DPZ in different groups from day 9 to 17 except the control group, to induce amnesia. On day 15, NOR was conducted 30 min after the administration of scopolamine and EPM was conducted on days 16 and 17 of the study. The animals were then sacrificed, and their whole brains were isolated for immunohistochemical and biochemical analyses.

4.5. Behavioral Analysis

4.5.1. Novel Object Recognition (NOR)

To assess the non-spatial and short-term memory analysis, the novel object recognition (NOR) test was used.47 The apparatus used for the NOR test consists of an open wooden box of the dimensions of 80 × 60 × 40 cm. For discrimination, two different shaped objects, one cylindrical and the other square, were used. The two objects were of similar height, which was approximately 10 cm from the lower surface. The whole study was divided into three phases: (i) habituation, (ii) familiarization, and (iii) retention. In the first phase, i.e., the habituation phase, the rats were allowed to move freely in the box for 15 min a day prior to trials. On the first day of the trial (T1), rats were allowed to assess two similar objects in the box (SO1 AND SO2) for 3 min. Rats were exposed to the second trial (T2) after 60 min of T1, and a novel object (NO) was introduced replacing one similar object. The time at which the rat’s nose was in proximity to the object was calculated as exploration time in the first and second trials separately. The formulation of the results was done by considering the discrimination of rats between SO and NO during T2. The equation for the calculation of the discrimination index is shown below.

4.5.1.

4.5.2. Elevated Plus Maze (EPM)

An elevated plus maze (EPM) device used for memory assessment consisted of four arms, two opposite open arms (50 × 10 cm) crossed with two closed arms with similar dimensions but with walls of 50 cm in height. The maze was 50 cm above the floor and had a central (10 × 10 cm) square giving the maze a plus shape, and the whole setup was placed in a dimly lit room.48 Rats were placed in the central area of the maze one at a time, and the number of entries in the closed and open arms was recorded; further time spent in the open and closed arms was also recorded. The whole experiment was conducted in two sessions for memory assessment; the first session (S1) was the training phase wherein rats were placed at the center of the device and were allowed to move in the open and closed arms. Using a stopwatch, transfer latency time (s) was noted, which is the time each rat takes to enter the open and closed arm. The session second (S2) was the retention phase; it was done 24 h after S1, and transfer latency time was noted. To explore the device, a cut-off time of 5 min was given to each rat. The index of memory improvement was calculated from a drop in latency.

4.6. Tissue Processing

For tissue processing, all the animals were anesthetized using mild anesthesia and sacrificed at the end of the experiment. Brains were isolated rapidly, cleaned free of unwanted materials, and perfused with ice-cold normal saline immediately. To perform immunohistochemical analysis, three brains from each group were preserved in 10% formalin immediately after washing, and hippocampi were isolated from the remaining rats for biochemical estimations.

4.7. Immunohistochemistry Studies

For the assessment of adult neurogenesis, BDNF and DCX were used on the hippocampus samples. For immunohistochemical analysis, three brain samples from each group were immersed in 4% paraformaldehyde and were kept overnight. The samples were further cryoprotected in sucrose for 24 h. In 15% polyvinylpyrrolidine, the brains were embedded and further frozen with dry ice and 40 μm frozen coronal sections using a cryostat. The storing of the sample sections was done with an anti-freeze buffer. Using 1% H2O2 in methanol, the sections were then quenched for 30 min. The sections were treated with a blocking buffer (1% BSA in PBS and 0.3% Triton X-100) for 1 h after washing with PBS. The sections were then incubated with BDNF and DCX antibodies overnight at 4 °C. The sections were incubated with secondary antibodies (Abcam) for 2 h after a second washing with PBS. Following the above process, the sections were treated with an avidin-biotin-peroxidase complex (Vectastin ABC kit, Vector) for 2 h. The activity of the samples was visualized using diaminobenzidine solution (DAB, Sigma). Using a microscope, the immunoreactions were monitored and results were calculated.

4.8. Homogenization

Hippocampi isolated from the rat brains were homogenized in PBS (0.1 M, pH 7.4) using a polytron homogenizer (Kinematica-AGPT 3000). The homogenate so prepared was filtered through a muslin cloth and was further centrifuged at 3000 rpm for 10 min at 4 °C in a Remi Cooling Centrifuge (C-24 DL) to separate the nuclear debris. For non-enzymatic assays, the aliquot so obtained was used as such, while to obtain the post-mitochondrial supernatant (PMS), it was further centrifuged at 10,000 rpm for 20 min at 4 °C, and the supernatant was used as a source for various enzyme assays.

4.9. Estimation of Different Biochemical Parameters

4.9.1. Estimation of Acetylcholinesterase Activity

For the determination of acetylcholinesterase (AChE) activity, a modified Ellman method was used.49,50 To a 100 μL buffered DTNB solution (10 mM DTNB and 15 mM sodium carbonate), 25 μL of acetylthiocholine iodide solution (75 mM) was added and kept at room temperature for 10 min. To start the enzymatic reaction, 10 μL of PMS was added to the test mixture and was incubated for 5 min. At 412 nm, the optical density (OD) was measured, and using a molar extinction coefficient (1.36 × 104 M–1 cm–1), AChE activity was calculated.

4.9.2. Estimation of Lipid Peroxidation (LPO)

Malondialdehyde (MDA) is the end product of membrane lipid peroxidation, and to quantify it, the modified method of Wills was used.51 Tissue homogenate (1 mL) was added to a mixture of 1.0 mL of TCA (10%) and 1.0 mL of TBA (0.67%) in each test tube. All the test tubes were then placed for a period of 20 min in a boiling water bath (95 °C). To cool down the reaction mixture, normal tap water was used. The extraction of the TBA-MDA product was done using 3 mL of n-butanol. The OD of the supernatant at 532 nm was noted, and the amount of MDA in each of the samples was calculated from it. To calculate the amount of MDA, a molar extinction coefficient of 1.56 × 105 M–1 cm–1 was used and the results so obtained were expressed as the nmol MDA formed/gram tissue.

4.9.3. Measurement of Superoxide Dismutase (SOD) Activity

For the determination of SOD activity, the Marklund method was used.52 The assay mixture (3 mL) was used, which consisted of 100 μL of PMS, 2.875 mL of Tris–Hcl buffer (50 mM, pH 8.5), and pyrogallol (24 mM in 10 Mm Hcl). The OD was recorded at 420 nm, and the enzyme activity was calculated from it and expressed as units/mg of protein. One unit was defined as the enzyme activity that inhibits auto-oxidation of 50% pyrogallol.

4.9.4. Measurement of Catalase (CAT) Activity

For the determination of catalase activity, the modified method of Claiborne was used.53 A total of 3 mL of the assay mixture was used for the assay consisting of 0.05 mL of PMS, 2.0 mL of PBS (0.1 M, pH 7.4), and 0.95 mL of hydrogen peroxide (0.020 M). OD was recorded at 240 nm and was used to calculate the catalase activity. The activity was calculated in the form of nmol H2O2 consumed/min/mg protein.

4.10. Measurement of Pro-Inflammatory Cytokine Levels

To estimate the pro-inflammatory cytokine levels (TNF-α, IL-1β, and IL-6) in the hippocampus,54 TNF-α, IL-1β, and IL-6 kits procured from Abbkine Scientific Co., Ltd. were used. The instructions in the manufacturer’s protocol were followed, which uses the principle of the sandwich ELISA technique for the evaluation of results. The experiment was started by adding 100 μL of the samples to each well in the assay plates and incubating them at room temperature for 2 h. The assay buffer from the kit was used to wash the plates, and TNF-α, IL-1β, and IL-6 conjugates were added to their specific wells and incubated for 1 h. The results were obtained from the absorbance at 450 nm, which was measured on a microplate reader.

5. Statistical Analysis

The data obtained from different groups were presented as the mean ± standard deviation (SD). GraphPad Prism v8.4 (GraphPad Software, San Diego, CA, United States) was used for all the statistical analysis. One-way ANOVA and Tukey multiple comparison tests in GraphPad Prism v8.4 were used for statistical analysis of variance between treated and control groups. For statistical significance, the minimum criterion was set at p < 0.05 for all the comparisons.

Acknowledgments

D.J.B. is a recipient of the Senior Research Fellowship from the Indian Council for Medical Research, Government of India [F.No. 45/48/2019-Nan/BMS].

The authors declare no competing financial interest.

Notes

Ethics approval was taken for this work.

Notes

Necessary consent was taken for publication.

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