The effect of zeolite zinc on memory performance and hippocampal cell death in a rat model of Alzheimer’s disease induced by Aβ1-42

doi:10.21203/rs.3.rs-4854823/v1

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The effect of zeolite zinc on memory performance and hippocampal cell death in a rat model of Alzheimer’s disease induced by Aβ_1-42

https://doi.org/10.21203/rs.3.rs-4854823/v1

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Alzheimer's disease (AD) is one of the most common neurodegenerative disorders associated with slow and progressive loss of brain structure and function mostly in older individuals. Evidence has shown that disruption of zinc homeostasis in the brain leads to synaptic, learning, and memory impairments. In this study, we evaluated the effect of zeolite zinc on memory performance and hippocampal cell death in a rat model of Alzheimer's disease (AD) induced by intracerebroventricular administration of Aβ1–42. We employed the Morris water maze, shuttle box, and open field tests to assess spatial memory, passive avoidance memory, and anxiety-like behavior, respectively.". P-Tau and the amyloid precursor protein (APP) expression, and hippocampal cell death were also evaluated. Both Aβ₁₋₄₂ and zeolite zinc were also injected intracerebroventricular. The results showed that zeolite zinc partially reversed Aβ1-42-induced impairments in memory performance and mitigated the effects of Aβ1–42 on locomotor activity, although not fully restoring to baseline levels. In addition, Aβ₁₋₄₂ increased the expression of APP and P-Tau, and the number of dead cells, while zeolite zinc decreased these effects. In conclusion, our findings suggest that while zeolite zinc plays a role in modulating the pathophysiology of AD, its therapeutic effects only partially reverse the progression or symptoms of AD, indicating the need for further investigation into optimal dosing or combination therapies

Aβ1−42

Zinc

Zeolite

Alzheimer’s disease

Memory

Hippocampus

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly population. AD is the leading cause of dementia, nearly 60–80% of all dementia cases in the world (1). AD is characterized by progressive neurodegeneration observed in both the cortex and the limbic system, along with the accumulation of β-amyloid (Aβ) and intraneuronal neurofibrillary tangles in the brain (2). Extracellular deposits of Aβ plaques and hyperphosphorylation of tau, a microtubule-associated protein induces intracellular formation of neurofibrillary tangles in the brain, are two major pathological hallmarks of AD (3). Of note, it has been shown that atherosclerosis (4), cerebrovascular endothelial damage (5), Aβ clearance dysfunction (6), oxidative damage following the blood–brain barrier (BBB) broken, and cerebral microvascular pathology (7, 8) are associated with the progression of AD. In AD, cognitive functions especially learning and memory are drastically impaired (9). Unfortunately, there is no an approved effective drug or treatment for AD and/or neurodegeneration, and related cognitive impairments (10).

Zinc can be a neuroprotective supplement. Zinc has important roles in suppressing inflammation and oxidative stress, and in the homeostasis of lipid and glucose metabolism particularly in the brain, with more concentration in the hippocampus and the amygdala (11, 12). Evidence shows that dietary and parenteral zinc supplementation decreases traumatic brain injury (TBI)-related depression and improves cognitive functions, particularly spatial learning and memory (13). On the contrary, excessive release of free zinc, especially in the hippocampus following acute injury, may potently increase protein ubiquitination and neural death (13). Prenatal zinc supplementation alleviates increased microglia and astrocyte density and increased inflammation following lipopolysaccharide administration in rats (14). It has been reported that post-weaning zinc supplementation can improve cognitive impairments induced by early life zinc deficiency in rat’s offspring (15). In addition, zinc partially restores obesity-related metabolic dysfunction and memory decline in rats (16). Previous study has shown that zinc reverses spatial memory impairment induced by streptozotocin-induced sporadic AD in rats (17). Also, co-treatment of zinc and selenium improves cognitive impairments, mitochondrial dysfunction, and oxidative stress in streptozotocin-induced sporadic AD in rats (18). However, as mentioned, zinc can induce opposite effects, such as increased oxidative stress, cognitive impairment, and tau phosphorylation (19).

In this study, we aim to investigate the effect of zinc zeolite on memory performance and hippocampal cell death in a rat model of AD induced by intracerebroventricular Aβ₁₋₄₂ administration.

2.1. Preparation of NaX/ZnO nanocomposite

The synthesis of NaX/ZnO nanocomposite involved two main stages. In the first stage, NaX zeolite nanocrystals were produced via a hydrothermal method. A unique mixture of aluminate and silicate solutions was prepared in a specific molar ratio to form aluminosilicate gel, using NaOH, NaAlO2, SiO2, and H2O. This gel was stirred continuously for half an hour at 27°C, followed by a hydrothermal treatment where the hydrogel was kept in a polypropylene bottle and agitated for 96 hours at 60°C at a consistent speed of 200 rpm. After this process, the material was isolated using centrifugation, thoroughly rinsed with de-ionized water to bring the pH below 8, and then dried at 70°C for 24 hours.

In the second stage, for preparing the NaX/ZnO nanocomposite with a ZnO weight percentage of 4.4%, a zinc nitrate solution of 0.05 molarity was initially made. Then, 1 gram of the nano-NaX zeolite powder was introduced into the solution. This suspension was then agitated vigorously at room temperature for four hours. The resulting powders were separated by centrifugation, washed with distilled water, and dried overnight at 110°C. Finally, the gathered products were calcined in air at 550°C for a duration of four hours.(20, 21).

2.2 Animals

Male Sprague-Dawley rats aged 8–10 weeks, weighing between 250–300 grams, were used in this study. The rats were sourced from a certified vendor to ensure uniform health and genetic background. They were housed under controlled conditions with a 12-hour light/dark cycle aligned with local time (lights on at 07:00 AM), at a temperature of 22 ± 2°C, and 40–60% humidity. Standard rodent chow and tap water were provided ad libitum. Environmental conditions were strictly monitored to maintain consistency throughout the study duration.

Prior to experimental testing, rats were acclimatized to the laboratory environment for one week, during which they were also habituated to handling to reduce stress related to experimental manipulations. All handling and testing were performed by trained personnel during the light phase of the cycle, specifically between 09:00 AM and 03:00 PM, to minimize circadian influences on behavioral outcomes. Rats were gently transported from the colony room to the testing room in their home cages to minimize stress. The transport process was designed to be quick and efficient, ensuring that the animals were not exposed to any additional stressors.

2.3 Experimental Design and Procedures

2.3.1 Testing Environment and Schedule

Behavioral assessments were conducted in a dedicated, sound-attenuated, and dimly lit testing room, separate from the colony room. This setup was designed to closely mimic the colony environment while providing a controlled setting for precise measurements. The testing room's light conditions were specifically adjusted to low levels to reduce anxiety and simulate the colony's conditions, ensuring behavioral consistency.

2.3.2 Testing Protocols and Hygiene

All behavioral tests, including the open field and shuttle box tests, were conducted at the same time each day to control for diurnal variations. The apparatus was cleaned thoroughly between each test subject to prevent scent marking and behavioral biases. A solution of 70% ethanol was used for cleaning, which effectively sanitizes the equipment without leaving residues that could affect animal behavior.

2.3.3 Euthanasia and Tissue Collection

At the conclusion of the behavioral tests, rats were euthanized via CO2 inhalation, following approved ethical guidelines. This method is recommended for its rapid onset and minimal stress to the animals. Following euthanasia, the brains were promptly perfused with phosphate-buffered saline to preserve tissue integrity for subsequent analyses. The hippocampus was then carefully dissected from each brain, focusing specifically on the right side for all animals to maintain consistency across samples. This selection was based on preliminary studies indicating right hemisphere dominance in spatial navigation tasks in rodents. Only four hippocampi per group were analyzed to maintain a manageable yet statistically significant sample size given the extensive histological and biochemical analyses required.

2.3.4 Personnel and Blinding Protocol

Experimental groups were coded, and the technicians were provided with the codes without knowledge of the group assignments. All data recording and analysis were conducted using these codes to ensure that the blinding was maintained throughout the study.Assessments were performed by two experienced technicians who were blinded to the experimental groupings of the animals. This blinding was essential to prevent observer bias in behavioral scoring and data interpretation. The technicians received comprehensive training on the specific protocols and scoring systems used in this study to ensure reliability and consistency of the data collected.

2.3.5 Experimental groups

This study consisted of four groups (n = 8). Control rats received saline. AD rats received Aβ1–42 at the dose of 5 µg/µL (2 µL was injected totally). AD + zinc zeolite

(i.c.v.) rats received both Aβ1–42 (at the mentioned dose, i.c.v.) and zinc zeolite at the dose of 1 µg/rat. AD + zinc zeolite

(i.p.) rats received both Aβ1–42 (at the mentioned dose, i.c.v.) and zinc zeolite at the dose of 3 mg/kg. At first, open field test was performed. Then, shuttle box and spatial memory tests were done, respectively.

2. Morris water maze (MWM) Testing Procedure

The Morris Water Maze (MWM) is a widely recognized experimental tool used to evaluate spatial learning and memory in rodents. This method is particularly effective in assessing the animals' abilities to recognize, recall, and navigate using spatial cues.

Description of the Maze:

The MWM setup in our study consisted of a dark-colored, circular tank measuring 150 cm in diameter and 60 cm in depth, filled with water maintained at a consistent temperature of 20 ± 2 ºC to a depth of 30 cm. The tank was segmented into four equal quadrants labeled as North (N), South (S), West (W), and East (E). Each quadrant had starting points positioned equidistantly along the tank’s perimeter. Various visual cues were placed on the walls surrounding the maze, and these remained unchanged throughout the experiment to serve as navigational aids for the rats.

Concealed Platform:

Central to the MWM experiment was a concealed platform, 10 cm in diameter, submerged 1 cm below the water's surface, located in the north-west quadrant. The movements of the rats within the maze were monitored by an overhead camera linked to a computer using the Etho-Vision XTv 8.5 video tracking system from Noldus Information Technology, Netherlands.

Testing Components:

The testing protocol included trial sessions and a probe test:

- Trial Sessions: Spanning four consecutive days, each day comprised four trials. Rats had 90 seconds to locate the hidden platform, using the wall cues for guidance. Upon finding the platform, they were allowed to remain there for 20 seconds to aid in the memorization of spatial cues. If a rat failed to locate the platform within the allotted time, it was gently guided there by the researcher and given a 20-second rest. Metrics recorded in each trial included escape latency (time taken to find the platform) and the distance traveled, both indicators of spatial learning efficiency.
- Probe Test: Conducted 24 hours after the last trial session, the hidden platform was removed for this 60-second test. The duration and distance that rats spent in the quadrant that previously contained the platform were measured, assessing their retention of spatial memory. Longer durations and distances in the target quadrant indicated better spatial memory retention.

Non-Spatial Visible Test:

Lastly, a non-spatial platform visible test was performed following the probe trial. Here, the platform was raised 2 cm above the water surface and covered with aluminum foil in the north-east quadrant. This test aimed to discern non-specific effects that might influence motor skills, vision, or motivational factors unrelated to learning and memory.

2.3. Shuttle box apparatus

The Shuttle Box apparatus, used for assessing passive avoidance memory, consists of two equal-sized compartments (25 × 25 × 25 cm each) — one illuminated (light compartment) and the other dark. Both compartments have a grid floor and are separated by a guillotine door.

Familiarization:

To familiarize the rats with the apparatus, each was placed in the shuttle box for a 5-minute session, 24 hours before the actual training. This pre-exposure helps to reduce anxiety and acclimatize the animals to the testing environment.

Training Session:

During the training session, each rat was placed in the light compartment for 60 seconds. After this period, the guillotine door was opened to allow the rat to move freely between the compartments. Upon entering the dark compartment, the door was closed, and a mild foot shock (0.5 mA, 50 Hz) was administered for 2 seconds through the grid floor. The training sessions, including the repeated exposure to the shock, were conducted on the same day. Between each of these exposures, a rest interval of 5 minutes was provided to allow the rat to recover and minimize stress, ensuring that the learning assessments were not influenced by excessive fatigue or stress."

Procedure for Non-Compliant Animals:

If during any trial a rat did not enter the dark compartment voluntarily after the door was opened, the experimenter gently nudged the animal towards it. This approach ensured that each rat received the intended training stimulus regardless of their initial reluctance. During the test session, if a rat consistently refused to enter the dark compartment, a maximum latency of 300 seconds was recorded. These occurrences were analyzed to differentiate between animals that learned to avoid the compartment due to memory of the shock and those that did not respond as expected.

Test Session:

The test session occurred 24 hours after the training session. Rats were again placed in the light compartment, and the step-through latency — the time taken for the rat to enter the dark compartment — was measured, up to a maximum of 300 seconds. This latency period is a measure of the rat's memory retention, as it indicates how well the rat remembers the shock associated with the dark compartment.

Handling Non-compliant Animals:

If a rat did not enter the dark compartment during the training session after the door was opened, it was gently nudged towards the dark compartment by the experimenter. This ensured that all animals received the intended training stimulus. If a rat consistently refused to enter the dark compartment during the test session, it was noted as a maximum latency (300 seconds) and analyzed accordingly. This procedure helps to include data from all animals, while clearly distinguishing between those that learned to avoid the dark compartment and those that did not respond as expected. (22, 23).

2.4. Open field test

The Open Field Test (OFT) was employed to evaluate locomotor activity and assess anxiety-like behavior in rats. For this test, we utilized a clear Perspex container measuring 100 x 100 cm with a height of 40 cm. The base of the arena was demarcated into 16 equal-sized squares to facilitate precise measurement of movement patterns.Each rat was individually placed at the center of the open field at the start of the test to ensure unbiased initial conditions. The session duration was set to 300 seconds (5 minutes), during which the rat's movements were continuously recorded. Locomotor activity was quantified by counting the number of squares crossed with all four paws, providing a direct measure of the rat's general activity level.Anxiety-like behavior was evaluated based on the time spent in the four central squares compared to the periphery. Rats spending more time near the walls, a behavior known as thigmotaxis, were considered to exhibit higher levels of anxiety. In contrast, more time spent in the center suggested reduced anxiety.The entire session was monitored using a camera positioned directly above the center of the field, ensuring a full aerial view. Movements within the arena were digitally tracked and analyzed using EthoVision XT software, which accurately measured the total distance traveled and the time spent in different zones of the field (24, 25)

2.5. Immunohistochemistry

Following the euthanasia of rats according to ethical protocols, brains were promptly harvested, and a 500 µm thick section of the hippocampus was prepared using a vibratome, focusing specifically on the CA1 region for detailed analysis. The immunohistochemistry process began with a series of washes in phosphate-buffered saline (PBS) to remove any debris and fixatives. Each wash was spaced five minutes apart to ensure thorough cleansing.

For antigen retrieval, hydrochloric acid was gently applied to the hippocampal sections for 30 minutes. To neutralize the acid, a borate buffer rinse followed for five minutes. The sections were then washed again with PBS to prepare for permeabilization and blocking. Cell membranes were permeabilized with 0.3% Triton X-100 for 30 minutes, enhancing antibody penetration. Subsequently, sections were incubated in 10% goat serum for another 30 minutes to block non-specific binding sites and minimize background staining.

Primary antibodies specific to the markers of interest were diluted at a ratio of 1:100 in PBS and applied to the sections. To prevent drying, the samples were placed in a humidified chamber and refrigerated at 2–8 degrees Celsius for 24 hours. After primary antibody incubation, sections underwent four PBS washes before application of the secondary antibody, diluted at 1:150. This antibody was incubated for 90 minutes at 37 degrees Celsius in a dark environment to prevent photobleaching.

Post-secondary antibody incubation, sections were washed in a dark room and subjected to DAPI staining to visualize nuclei. This was followed by a final PBS rinse to remove excess DAPI.

Image Analysis:

Immunostained sections were examined under an Olympus fluorescent microscope at 400x magnification. Digital images of the stained samples were captured for analysis. Image analysis was performed using specialized software (ImageJ), which allows for the quantification of staining intensity and the distribution of immunoreactive cells within the hippocampal CA1 region.

Quantitative analysis involved measuring the intensity of staining and counting the number of positive cells per unit area. These measurements were used to assess the relative expression of the target proteins in different experimental groups. Qualitative assessments were also made, noting the localization and morphological characteristics of the staining to provide insights into the pathological changes in the hippocampus. Results from the image analysis were statistically analyzed to determine significant differences between experimental groups, providing insights into the effects of the experimental treatments on hippocampal structure and function.

2.6. Statistical analyses

Statistical analyses in the study were conducted using SPSS software, version 26.0. To determine significant differences between the groups, a variety of statistical tests were employed. These included repeated measure analysis and one-way ANOVA, followed by post hoc Tukey’s tests for detailed comparison. In assessing the results, a p-value of less than 0.05 was established as the threshold for statistical significance. This approach ensured a comprehensive and rigorous examination of the data, allowing for precise interpretation of the differences observed among the various experimental groups.

Results

2.7. Spatial Learning and Spatial Memory from Morris Water Maze Tests

Training- Repeated measure analysis revealed that the effect of training [For escape latency-(F_3,84 = 48.97, P < 0.001) and the effect of training*group [(F_9.84 = 84.83, P < 0.001); [For traveled distance-(F_3,84 = 33.07, P < 0.001) and the effect of training*group [(F_9.84 = 3.50, P < 0.001) were significant. Post hoc Tukey test also showed that administration of Aβ₁₋₄₂ increased escape latency and traveled distance (P < 0.001; training day 2–4), meaning impaired spatial learning. However, zeolite zinc (only i.c.v. injection, P < 0.001, training day 2–4) reversed the impairment effect of Aβ₁₋₄₂ on spatial learning in AD rats (Fig. 1).

Probe- The results of one-way ANOVA showed that there was a significant difference between groups for time spent (F_3.28 = 28.04, P < 0.001) and traveled distance (F_3.28 = 52.92, P < 0.001). Post hoc Tukey test showed that AD rats spent less time (P < 0.001) and distance (P < 0.01) in the target quadrant, meaning spatial memory performance was impaired in AD rats. However, zeolite zinc (only i.c.v. injection, for time spent: P < 0.001, for traveled distance: P < 0.01) reversed this effect (Fig. 2).

Figure 1 and 2 here

2.8. Passive Avoidance Memory from Shuttle Box Tests

The results of one-way ANOVA showed that there was a significant difference between groups (F_3.28 = 3.68, P < 0.001). Post hoc Tukey test showed that AD rats had lesser latency to enter dark chamber (P < 0.001), meaning passive avoidance memory impairment. However, zeolite zinc (i.c.v., P < 0.001; i.p., P < 0.01) reversed this effect (Fig. 3).

Figure 3 here

2.9. Locomotor Activity and Anxiety-like Behavior from Open Field Tests

The results of one-way ANOVA showed that there was a significant difference between groups for total distance (F_3.28 = 7.69, P < 0.001). Post hoc Tukey test showed that AD rats had slightly lesser locomotor activity (P < 0.05). However, zeolite zinc (i.c.v. and i.p., P < 0.05) reversed this effect. The results of one-way ANOVA showed that there was a significant difference between groups for time spent in the middle squares (F_3.28 = 21.72, P < 0.001). Post hoc Tukey test showed that AD rats spent less time in the middle squares (P < 0.001), suggesting anxiety-like behavior. However, zeolite zinc (i.c.v., P < 0.001; i.p., P < 0.01) reversed this effect (Fig. 4).

Figure 4 here

2.10. APP and P-Tau Expression and Cell Viability from Immunohistochemical Analyses

The results of one-way ANOVA showed that there was a significant difference between groups for APP expression (F_3.12 = 412.52, P < 0.001). Post hoc Tukey test showed that APP expression was increased in AD (P < 0.001). Furthermore, zeolite zinc (i.c.v., P < 0.001; i.p., P < 0.01) reversed this effect. The results of one-way ANOVA showed that there was a significant difference between groups for P-Tau expression (F_3.28 = 255.21, P < 0.001). Post hoc Tukey test showed that P-Tau expression was increased in AD (P < 0.001). Furthermore, zeolite zinc (i.c.v., P < 0.001; i.p., P < 0.01) reversed this effect. The results of one-way ANOVA showed that there was a significant difference between groups for dead cells counting (F_3.28 = 235.30, P < 0.001). Post hoc Tukey test showed that dead cells was increased in AD rats (P < 0.001). Furthermore, zeolite zinc (i.c.v., P < 0.001; i.p., P < 0.01) reversed this effect. Note that, zeolite zinc (i.c.v.) did not completely restore the effect of Aβ₁₋₄₂ administration on APP, P-Tau, and dead cells, and showed a significant difference in comparison with controls (P < 0.05: for APP and dead cells, P < 0.01: for P-Tau). Also, zeolite zinc (i.p.) did not completely restore the effect of Aβ₁₋₄₂ administration on APP, P-Tau, and dead cells, and showed a significant difference in comparison with controls (P < 0.001 for all) (Fig. 5).

Figure 5 here

Discussion

2.11. Overview

As the results demonstrated, zeolite zinc effectively reversed spatial learning and memory impairments (observed only with intracerebroventricular (i.c.v.) injection) and passive avoidance memory deficits (seen with both i.c.v. and intraperitoneal (i.p.) injections) induced by Aβ1–42 administration. In the open field test, not only did zeolite zinc mitigate the impact of Aβ1–42 on locomotor activity, but it also significantly reduced anxiety-like behaviors, as evidenced by increased time spent in the center of the arena compared to the periphery. Additionally, Aβ1–42 administration was associated with increased expression of amyloid precursor protein (APP) and phosphorylated Tau (P-Tau), as well as an increase in the number of dead cells. Conversely, zeolite zinc administration decreased these pathological effects, highlighting its potential neuroprotective properties.

2.12. Aβ_1-42 administration and memory impairment

As we know, the most important pathological feature of AD is memory impairments. Administration of Aβ₁₋₄₂ in rodents can induce an AD model, because abnormal accumulation of Aβ_1–42 is considered as a causative agent in the pathology of AD (26). Also, past studies have shown that Aβ_1–42 has adverse effects on neural functions in vitro (27, 28). Previous reports have shown that chronic increase in brain Aβ leads to cognitive deficits, and in vitro studies show extensive neurotoxicity of Aβ (29–31). Neurotoxic Aβ_1–42 oligomers seem to act, at least partly, by the prevention of insulin signaling that promotes synaptic functions (28, 32). Note that, Aβ_1–42 oligomers may acutely impair both cognitive and metabolic functions within the hippocampus, which appears to be mediated via interference with insulin-regulated glucose transport and metabolism, a main modulator of the function of the hippocampus (33). In addition, injection of Aβ neuronal can induce apoptosis. Previous research has shown that intracranial injection of Aβ leads to cell death and apoptosis in the hippocampus of rats (34). Furthermore, it has been shown that the neurotoxicity mechanism of Aβ is related to oxidative stress and apoptosis, as shown by enhanced malonaldehyde generation and TUNEL-positive cells (35). It has also been shown that Aβ₁₋₄₂ induces spatial memory and learning dysfunction, and leads to neuronal apoptosis and autophagy in the hippocampus (36). Aβ can impair long-term potentiation (LTP) induction at the PP-DG hippocampal synapses, evidenced by a decrease in excitatory postsynaptic potentials (EPSP) slope and population spike (PS) amplitude of LTP (37). Our data also showed that Aβ₁₋₄₂ administration impaired spatial and passive avoidance memory performance in rats.

2.13. The effect of zinc on memory function

Furthermore, our data showed that intracerebroventricular administration of zeolite zinc reversed memory impairment induced by Aβ₁₋₄₂. Zinc released from hippocampal vesicles can produce a broad spectrum of neuromodulatory effects upon target cells. Zinc-containing hippocampal neurons have been found predominantly in limbic and cerebrocortical areas, with a role in the modification of synaptic strength (38). Importantly, vesicular zinc-enriched regions such as the hippocampus are highly susceptible to dietary zinc deprivation, which may lead to cognitive impairments such as learning and memory impairment, and olfactory dysfunction (39). Previous reports have shown that zinc administration induces a protective effect on different neurodegenerative models, including ischemic brain injury or AD (40, 41). As we know, zinc is an essential trace element obtained from the diet that regulates the expression and activation of many biological molecules. Zinc is a critical micronutrient that plays an essential role in a vast number of physiological processes, and it should be noted that, about 10% of proteins have zinc binding domains (42). Surprisingly, more than 2 billion people worldwide have zinc deficiency, while it has profound effects on the function of the immune system (43). Importantly, zinc deficiency appears to have a close association with AD, which can contribute to poorer quality of life and disease outcome (40). It has been shown that zinc deficiency and AD has a close relationship, due to the reports shown the reduced plasma zinc level in individuals with AD (44). It has been revealed that AD patients have a significant decrease in CSF zinc but not serum zinc level (45). Decreased serum zinc level has also been shown, while some AD patients enrolled in one study appeared to be malnourished (46, 47). Zinc deficiency also leads to abnormal glucocorticoid secretion and increases depression-like behavior (48), which can also lead to memory impairment. Also, reduced brain zinc availability decreases hippocampal neurogenesis in mice and rats (49).

On the other hand, a redistribution of zinc may be sufficient to promote disease progression (50). Note that, the most prominent brain lesions in patients with AD are the amyloid or “senile” plaques, which predominantly include Aβ peptides derived from the proteolytic processing of the amyloid precursor protein (APP) (50). As we know, APP is processed by one of two pathways: the amyloidogenic pathway that eventually produces Aβ, and the non-amyloidogenic pathway (51). Amyloid plaques have been reported to contain higher concentrations of copper, iron, and zinc (52, 53). Previous study has shown higher concentration of zinc (up to 1 mM) within amyloid plaques (54), that may be released from glutamatergic synapses (55). Therefore, Aβ aggregation may be rapidly induced in the presence of zinc ions under physiological condition in vitro (56). In addition, previous study has suggested that zinc plays an important role in Aβ aggregation, which is well established that zinc may play a role in the pathogenesis of AD (50). Also, synaptic zinc promotes Aβ oligomer formation and their accumulation at excitatory synapses (57). Elevated levels of zinc in the hippocampus and the amygdala of AD patients have also been shown (58). Previous study has shown that low-dose zinc supplementation in obese mice can restore the high-fat diet-induced reduction in neurogenic and synaptic marker proteins in the hippocampus via decreasing lipid peroxidation and improving BDNF expression, while high-dose zinc supplementation exacerbates the reduction of neurogenesis via affecting synaptic markers and BDNF levels in the hippocampus (59). In sum, it seems that both zinc deficiency, or increased zinc levels may be related to the pathophysiology of AD. In the present study, administration of zeolite zinc partially reversed memory impairment induced by Aβ1–42. This effect may be attributed to zinc’s known role in modulating synaptic plasticity and neurotransmission. Zinc ions can modulate glutamatergic activity which is crucial for learning and memory processes. Furthermore, zinc possesses antioxidant properties that may mitigate oxidative stress associated with Aβ pathology in Alzheimer's disease. We also observed a reduction in anxiety-like behaviors, which could be linked to zinc's influence on the central GABAergic system, known for regulating anxiety states in neurological disorders

2.14. The effect of zinc on cell death and apoptosis

Additionally, our data showed that zeolite zinc decreased cell death in AD rats. The immune system, especially inflammation, is highly susceptible to the changes of zinc levels (60). It has been shown that zinc homeostasis affects neuroinflammation in the brain (17), that can also affect cognitive functions. Also, zinc is a potent inhibitor of apoptosis, while zinc depletion induces apoptosis in many cell lines (61). Changes in intracellular zinc concentrations have a critical role in modulating apoptosis (62). It has been shown that the prevention of apoptosis by zinc is associated with the inhibition of an endonuclease acting in the late phase of apoptosis (63). A wide-range of studies have shown that zinc deficiency has been implicated in apoptosis in both in vitro and in vivo models (64, 65). Furthermore, zinc deficiency decreases neurogenesis accompanied by neural apoptosis via caspase-dependent and -independent signaling pathways (66). It has been shown that depletion of intracellular zinc leads to apoptosis of cultured hippocampal neurons via suppressing ERK signaling pathway and activating caspase-3 (67). Previous research has also shown that zinc restored memory impairment induced by streptozotocin via affecting proteins involved in hippocampal synaptic plasticity (17). Furthermore, zinc deficiency leads to apoptotic neural cell death via the intrinsic mitochondrial pathway, triggered by activating caspase-3 and abnormal modulation of pro-survival pathways (extracellular signal-regulated kinase (ERK1/2), nuclear factor-κB (NF-κB)) (68, 69).

Interestingly, it has been shown that zinc can induce a dose-dependent effect on apoptosis. Previous research has shown that high zinc concentrations (from 600 to 75 microM) inhibit apoptosis, whereas low zinc concentrations (from 15 to 7.5 microM) induce apoptosis or increase serum-free medium-induced apoptosis (70). Also, the pro-apoptotic effects of zinc have been revealed in previous studies (71). It has been shown that zinc, even at pharmacologic concentrations, affects cytokine expression and induces apoptosis of human peripheral blood mononuclear cells (72). In addition, excessive zinc accumulation following cerebral ischemia can activate inflammatory mediators and inflammation-mediated neuronal apoptosis (73). In sum, it seems that zinc has a dual effect on cell death and apoptosis. However, our data showed that intracerebroventricular injection of zeolite zinc decreases cell death induced by Aβ₁₋₄₂. In this study, we observed that zeolite zinc, when administered intracerebroventricularly, effectively ameliorated spatial learning and memory deficits induced by Aβ1–42. This observation suggests that zinc may interact with key neural pathways affected by Alzheimer's pathology. Specifically, zinc is known to play a crucial role in enhancing synaptic plasticity and neurotransmission, which are vital for memory and learning processes. Research indicates that zinc modulates the activity of glutamate receptors and may help stabilize synaptic functions, which are often disrupted in the context of Alzheimer's disease (55).

Moreover, the antioxidant properties of zinc contribute to its therapeutic effects. Oxidative stress is a hallmark of Alzheimer's disease, leading to neuronal damage and loss of cognitive function. Zinc's ability to reduce oxidative stress—by scavenging reactive oxygen species and enhancing the antioxidant defense system of neurons—provides a plausible explanation for its neuroprotective effects observed in our study (11, 12).

Additionally, the reduction in anxiety-like behaviors seen in the open field test, with rats spending more time in the center of the arena, underscores zinc's potential influence on anxiety regulation mechanisms. This effect might be mediated through zinc's modulation of the GABAergic system, a key pathway involved in anxiety and mood regulation(74). Considering our study's constraints, it is advisable for subsequent research to incorporate a range of dosage levels and establish distinct control groups for each administration method. This approach will enable a more detailed analysis of dose-response relationships and the relative efficacy of various delivery methods. Expanding the experimental framework in this way will enhance our understanding of how zeolite zinc influences Alzheimer's disease pathology, allowing for a more comprehensive assessment of its therapeutic mechanisms and potential.

Conclusion

In conclusion, the results of the present study showed that zeolite zinc reversed spatial learning and memory impairment (i.c.v. injection), and passive avoidance memory deficit (i.c.v. and i.p. injections) induced by Aβ₁₋₄₂ administration. In the open field test, zeolite zinc reversed the effect of Aβ₁₋₄₂ administration on locomotor activity. Furthermore, Aβ₁₋₄₂ administration increased the expression of APP and P-Tau, and the number of dead cells, while zeolite zinc decreased these effects. Although zinc may show a dose-dependent or dual effect on both memory function and cell death, in the present research, zeolite zinc partially reversed memory impairment and decreased cell death in AD rats.

Ethical Considerations

Ethical clearance was secured from Research Ethics Committees of Tehran Islamic Azad University Of Medical Sciences, Iran, under the reference numbers IR.IAU.TMU.REC.1401.063 and IR.IAU.PS.REC.1402.036. The research adhered to all relevant international, national, and institutional guidelines for the ethical treatment of animals.

Participant Consent

Not required for this research.

Publication Consent

Due to privacy and confidentiality concerns, the data from this study are confidential and not publicly accessible. The principal investigator may release the data in response to substantiated inquiries.

3.1. Spatial Learning and Spatial Memory from Morris Water Maze Tests

Training- Repeated measure analysis revealed that the effect of training [For escape latency-(F_3,84= 48.97, P < 0.001) and the effect of training*group [(F_9.84 = 84.83, P < 0.001); [For traveled distance-(F_3,84= 33.07, P < 0.001) and the effect of training*group [(F_9.84 = 3.50, P < 0.001) were significant. Post hoc Tukey test also showed that administration of Aβ_1-42increased escape latency and traveled distance (P<0.001; training day 2-4), meaning impaired spatial learning. However, zeolite zinc (only i.c.v. injection, P<0.001, training day 2-4) reversed the impairment effect of Aβ_1-42on spatial learning in AD rats (Fig.1).

Probe- The results of one-way ANOVA showed that there was a significant difference between groups for time spent (F_3.28 = 28.04, P<0.001) and traveled distance (F_3.28 = 52.92, P<0.001). Post hoc Tukey test showed that AD rats spent less time (P<0.001) and distance (P<0.01) in the target quadrant, meaning spatial memory performance was impaired in AD rats. However, zeolite zinc (only i.c.v. injection, for time spent: P<0.001, for traveled distance: P<0.01) reversed this effect (Fig.2).

Fig.1 and 2 here

3.2. Passive Avoidance Memory from Shuttle Box Tests

The results of one-way ANOVA showed that there was a significant difference between groups (F_3.28 = 3.68, P<0.001). Post hoc Tukey test showed that AD rats had lesser latency to enter dark chamber (P<0.001), meaning passive avoidance memory impairment. However, zeolite zinc (i.c.v., P<0.001; i.p., P<0.01) reversed this effect (Fig.3).

Fig.3 here

3.3. Locomotor Activity and Anxiety-like Behavior from Open Field Tests

The results of one-way ANOVA showed that there was a significant difference between groups for total distance (F_3.28 = 7.69, P<0.001). Post hoc Tukey test showed that AD rats had slightly lesser locomotor activity (P<0.05). However, zeolite zinc (i.c.v. and i.p., P<0.05) reversed this effect. The results of one-way ANOVA showed that there was a significant difference between groups for time spent in the middle squares (F_3.28 = 21.72, P<0.001). Post hoc Tukey test showed that AD rats spent less time in the middle squares (P<0.001), suggesting anxiety-like behavior. However, zeolite zinc (i.c.v., P<0.001; i.p., P<0.01) reversed this effect (Fig.4).

Fig.4 here

3.4. APP and P-Tau Expression and Cell Viability from Immunohistochemical Analyses

The results of one-way ANOVA showed that there was a significant difference between groups for APP expression (F_3.12 = 412.52, P<0.001). Post hoc Tukey test showed that APP expression was increased in AD (P<0.001). Furthermore, zeolite zinc (i.c.v., P<0.001; i.p., P<0.01) reversed this effect. The results of one-way ANOVA showed that there was a significant difference between groups for P-Tau expression (F_3.28 = 255.21, P<0.001). Post hoc Tukey test showed that P-Tau expression was increased in AD (P<0.001). Furthermore, zeolite zinc (i.c.v., P<0.001; i.p., P<0.01) reversed this effect. The results of one-way ANOVA showed that there was a significant difference between groups for dead cells counting (F_3.28 = 235.30, P<0.001). Post hoc Tukey test showed that dead cells was increased in AD rats (P<0.001). Furthermore, zeolite zinc (i.c.v., P<0.001; i.p., P<0.01) reversed this effect. Note that, zeolite zinc (i.c.v.) did not completely restore the effect of Aβ_1-42administration on APP, P-Tau, and dead cells, and showed a significant difference in comparison with controls (P<0.05: for APP and dead cells, P<0.01: for P-Tau). Also, zeolite zinc (i.p.) did not completely restore the effect of Aβ_1-42administration on APP, P-Tau, and dead cells, and showed a significant difference in comparison with controls (P<0.001 for all) (Fig.5).

Fig.5 here

4.1. Overview

As the results demonstrated, zeolite zinc effectively reversed spatial learning and memory impairments (observed only with intracerebroventricular (i.c.v.) injection) and passive avoidance memory deficits (seen with both i.c.v. and intraperitoneal (i.p.) injections) induced by Aβ1-42 administration. In the open field test, not only did zeolite zinc mitigate the impact of Aβ1-42 on locomotor activity, but it also significantly reduced anxiety-like behaviors, as evidenced by increased time spent in the center of the arena compared to the periphery. Additionally, Aβ1-42 administration was associated with increased expression of amyloid precursor protein (APP) and phosphorylated Tau (P-Tau), as well as an increase in the number of dead cells. Conversely, zeolite zinc administration decreased these pathological effects, highlighting its potential neuroprotective properties.

4.2. Aβ_1-42administration and memory impairment

As we know, the most important pathological feature of AD is memory impairments. Administration of Aβ_1-42in rodents can induce an AD model, because abnormal accumulation of Aβ_1–42is considered as a causative agent in the pathology of AD (26). Also, past studies have shown that Aβ_1–42 has adverse effects on neural functions in vitro (27, 28). Previous reports have shown that chronic increase in brain Aβ leads to cognitive deficits, and in vitro studies show extensive neurotoxicity of Aβ (29-31). Neurotoxic Aβ_1–42 oligomers seem to act, at least partly, by the prevention of insulin signaling that promotes synaptic functions (28, 32). Note that, Aβ_1–42oligomers may acutely impair both cognitive and metabolic functions within the hippocampus, which appears to be mediated via interference with insulin-regulated glucose transport and metabolism, a main modulator of the function of the hippocampus (33). In addition, injection of Aβ neuronal can induce apoptosis. Previous research has shown that intracranial injection of Aβ leads to cell death and apoptosis in the hippocampus of rats (34). Furthermore, it has been shown that the neurotoxicity mechanism of Aβ is related to oxidative stress and apoptosis, as shown by enhanced malonaldehyde generation and TUNEL-positive cells (35). It has also been shown that Aβ_1-42 induces spatial memory and learning dysfunction, and leads to neuronal apoptosis and autophagy in the hippocampus (36). Aβ can impair long-term potentiation (LTP) induction at the PP-DG hippocampal synapses, evidenced by a decrease in excitatory postsynaptic potentials (EPSP) slope and population spike (PS) amplitude of LTP (37). Our data also showed that Aβ_1-42administration impaired spatial and passive avoidance memory performance in rats.

4.3. The effect of zinc on memory function

Furthermore, our data showed that intracerebroventricular administration of zeolite zinc reversed memory impairment induced by Aβ_1-42. Zinc released from hippocampal vesicles can produce a broad spectrum of neuromodulatory effects upon target cells. Zinc-containing hippocampal neurons have been found predominantly in limbic and cerebrocortical areas, with a role in the modification of synaptic strength (38). Importantly, vesicular zinc-enriched regions such as the hippocampus are highly susceptible to dietary zinc deprivation, which may lead to cognitive impairments such as learning and memory impairment, and olfactory dysfunction (39). Previous reports have shown that zinc administration induces a protective effect on different neurodegenerative models, including ischemic brain injury or AD (40, 41). As we know, zinc is an essential trace element obtained from the diet that regulates the expression and activation of many biological molecules. Zinc is a critical micronutrient that plays an essential role in a vast number of physiological processes, and it should be noted that, about 10% of proteins have zinc binding domains (42). Surprisingly, more than 2 billion people worldwide have zinc deficiency, while it has profound effects on the function of the immune system (43). Importantly, zinc deficiency appears to have a close association with AD, which can contribute to poorer quality of life and disease outcome (40). It has been shown that zinc deficiency and AD has a close relationship, due to the reports shown the reduced plasma zinc level in individuals with AD (44). It has been revealed that AD patients have a significant decrease in CSF zinc but not serum zinc level (45). Decreased serum zinc level has also been shown, while some AD patients enrolled in one study appeared to be malnourished (46, 47). Zinc deficiency also leads to abnormal glucocorticoid secretion and increases depression-like behavior (48), which can also lead to memory impairment. Also, reduced brain zinc availability decreases hippocampal neurogenesis in mice and rats (49).

On the other hand, a redistribution of zinc may be sufficient to promote disease progression (50). Note that, the most prominent brain lesions in patients with AD are the amyloid or “senile” plaques, which predominantly include Aβ peptides derived from the proteolytic processing of the amyloid precursor protein (APP) (50). As we know, APP is processed by one of two pathways: the amyloidogenic pathway that eventually produces Aβ, and the non-amyloidogenic pathway (51). Amyloid plaques have been reported to contain higher concentrations of copper, iron, and zinc (52, 53). Previous study has shown higher concentration of zinc (up to 1 mM) within amyloid plaques (54), that may be released from glutamatergic synapses (55). Therefore, Aβ aggregation may be rapidly induced in the presence of zinc ions under physiological condition in vitro (56). In addition, previous study has suggested that zinc plays an important role in Aβ aggregation, which is well established that zinc may play a role in the pathogenesis of AD (50). Also, synaptic zinc promotes Aβ oligomer formation and their accumulation at excitatory synapses (57). Elevated levels of zinc in the hippocampus and the amygdala of AD patients have also been shown (58). Previous study has shown that low-dose zinc supplementation in obese mice can restore the high-fat diet-induced reduction in neurogenic and synaptic marker proteins in the hippocampus via decreasing lipid peroxidation and improving BDNF expression, while high-dose zinc supplementation exacerbates the reduction of neurogenesis via affecting synaptic markers and BDNF levels in the hippocampus (59). In sum, it seems that both zinc deficiency, or increased zinc levels may be related to the pathophysiology of AD. In the present study, administration of zeolite zinc partially reversed memory impairment induced by Aβ1-42. This effect may be attributed to zinc’s known role in modulating synaptic plasticity and neurotransmission. Zinc ions can modulate glutamatergic activity which is crucial for learning and memory processes. Furthermore, zinc possesses antioxidant properties that may mitigate oxidative stress associated with Aβ pathology in Alzheimer's disease. We also observed a reduction in anxiety-like behaviors, which could be linked to zinc's influence on the central GABAergic system, known for regulating anxiety states in neurological disorders

4.4. The effect of zinc on cell death and apoptosis

Interestingly, it has been shown that zinc can induce a dose-dependent effect on apoptosis. Previous research has shown that high zinc concentrations (from 600 to 75 microM) inhibit apoptosis, whereas low zinc concentrations (from 15 to 7.5 microM) induce apoptosis or increase serum-free medium-induced apoptosis (70). Also, the pro-apoptotic effects of zinc have been revealed in previous studies (71). It has been shown that zinc, even at pharmacologic concentrations, affects cytokine expression and induces apoptosis of human peripheral blood mononuclear cells (72). In addition, excessive zinc accumulation following cerebral ischemia can activate inflammatory mediators and inflammation-mediated neuronal apoptosis (73). In sum, it seems that zinc has a dual effect on cell death and apoptosis. However, our data showed that intracerebroventricular injection of zeolite zinc decreases cell death induced by Aβ_1-42. In this study, we observed that zeolite zinc, when administered intracerebroventricularly, effectively ameliorated spatial learning and memory deficits induced by Aβ1-42. This observation suggests that zinc may interact with key neural pathways affected by Alzheimer's pathology. Specifically, zinc is known to play a crucial role in enhancing synaptic plasticity and neurotransmission, which are vital for memory and learning processes. Research indicates that zinc modulates the activity of glutamate receptors and may help stabilize synaptic functions, which are often disrupted in the context of Alzheimer's disease (55).

In conclusion, the results of the present study showed that zeolite zinc reversed spatial learning and memory impairment (i.c.v. injection), and passive avoidance memory deficit (i.c.v. and i.p. injections) induced by Aβ_1-42administration. In the open field test, zeolite zinc reversed the effect of Aβ_1-42administration on locomotor activity. Furthermore, Aβ_1-42administration increased the expression of APP and P-Tau, and the number of dead cells, while zeolite zinc decreased these effects. Although zinc may show a dose-dependent or dual effect on both memory function and cell death, in the present research, zeolite zinc partially reversed memory impairment and decreased cell death in AD rats.

Ethical Considerations: Ethical clearance was secured from Research Ethics Committees of Tehran Islamic Azad University Of Medical Sciences, Iran, under the reference numbers IR.IAU.TMU.REC.1401.063 and IR.IAU.PS.REC.1402.036. The research adhered to all relevant international, national, and institutional guidelines for the ethical treatment of animals.

Participant Consent: Not required for this research.

Publication Consent: Due to privacy and confidentiality concerns, the data from this study are confidential and not publicly accessible. The principal investigator may release the data in response to substantiated inquiries.

Conflict of Interest: No conflicts of interest were declared by any authors of this study.

Author Contributions: MZF and AB formulated the original idea and oversaw the study's implementation. H.R.SH played a pivotal role in synthesizing the NaX/ZnO nanocomposite. SV, FKH, and FR significantly contributed to designing the study's framework, executing operations, and refining the manuscript. BGY was instrumental in developing the research concept, planning experiments, data analysis, and manuscript revision. All authors have reviewed and consented to the manuscript's publication.

Financial Support: The study did not receive any specific financial support.

Data Accessibility: Data from this research are available upon a well-founded request.

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No competing interests reported.