Effect of JM-20 on Age-related cognitive impairment in mice

doi:10.21203/rs.3.rs-4863448/v1

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Effect of JM-20 on Age-related cognitive impairment in mice

https://doi.org/10.21203/rs.3.rs-4863448/v1

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The decline in cognitive function associated with aging significantly impacts the well-being of elderly individuals and their families. This decline is a major recognized risk factor for neurodegenerative diseases, notably Alzheimer's disease. Animal models of aging provide a platform for evaluating drugs concerning aspects like memory and oxidative stress. JM-20 has demonstrated protective effects on short-term memory acquisition and consolidation, along with antioxidant properties and modulation of Acetylcholinesterase activity. This study assesses the potential protective JM-20 against cognitive decline and age-related memory loss. For the study, aged mice exhibiting aging-associated damage were initially selected. Experimental groups were then formed, and the effect of 8 mg/kg of JM-20 was evaluated for 40 days on aging-related behavior, such as spatial memory, novelty recognition memory, ambulatory activity, and anxiety. Subsequently, animals were sacrificed, and the hippocampal region was extracted for redox studies and to assess acetylcholinesterase activity. Results indicated that JM-20 at 8 mg/kg reversed damage to spatial working and reference memory, exhibiting performance comparable to untreated young adult animals. Furthermore, JM-20 preserved the enzymatic activity of superoxide dismutase, catalase, and total sulfhydryl levels in age-related cognitive impairment in mice, indicating a potent protective effect against oxidative events at the brain level. However, only young, healthy animals showed decreased acetylcholinesterase enzyme activity. These findings provide preclinical pharmacological evidence supporting the neuroprotective activity of JM-20, positioning it as a promising therapeutic candidate for treating memory disorders associated with aging.

Age-related cognitive impairment in mice

dementia

JM-20

neuroprotection

Aging represents the gradual decline of time-dependent physiological integrity, leading to an increased susceptibility to mortality [1]. It stands as the predominant recognized risk factor for the onset of neurodegenerative diseases, including the most prevalent form, Alzheimer's disease [2, 3]. Clinical manifestations of aging encompass cognitive impairment, memory lapses, reduced information processing speed, depression, anxiety, muscle weakness, compromised motor functions, urinary incontinence, and ulcerations [4, 5]. Consequently, independence in performing daily activities becomes constrained.

Genomic instability is a hallmark of the aging process, marked by telomere attrition, epigenetic modifications, and loss of proteostasis. Simultaneously, antagonistic features emerge, such as mitochondrial dysfunction, cellular senescence, nutrient impairment, and the generation of free radicals, initially serving as compensatory responses to primary deterioration. However, when sustained over time, these antagonistic characteristics become deleterious [1, 6].

Models utilizing young and aged rodents have been employed to investigate age-related behavioral changes. These studies revealed that aged mice exhibit impairments in sensory, motor, and cognitive functions. Accordingly, there is a decline in behavioral performance and learning abilities [7, 8]. Additionally, aged mice may display signs indicative of anxiety and depression. Animal models involving aged rodents has to successfully induce cognitive deficits reminiscent of clinical occurrences [9, 10]. Therefore, the validated model is well-suited for cognitive impairments is well-suited for evaluating the efficacy of novel candidates for the treatment or to prevent diseases associated with cognitive and memory impairments. For this reason, the evaluation of the novel synthetic hybrid molecule JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1, 5]-benzodiazepine) is of interest.

In past experiments, JM-20 demonstrated a potent protective effect on memory, improving the processes of short-term memory acquisition and consolidation. It has an antioxidant effect, protects mitochondrial function, and modulates cholinesterase’s (ChEs) activities [11–13]. JM-20 also preserved anti-apoptotic proteins and neural integrity in hippocampal and prefrontal cortex regions [12]. Therefore, here we evaluated the potential protective effect of JM-20 against cognitive decline and memory loss associated with aging in mice.

Compounds and reagents

Unless otherwise specified, all chemicals used were of the highest grade available and purchased from Sigma-Aldrich (St. Louis, MO, USA). JM-20 was synthesized, purified, and characterized as previously reported [14].

Experimental animals

Male C57BL/6 mice aged (14–16 months old, weighing 30–35 g) and young adults (8–9 weeks, weighing 22–25 g) were obtained from the National Center for the Production of Laboratory Animals (CENPALAB, Mayabeque, Cuba). Mice were maintained in the room under conventional laboratory conditions (12 h light/dark cycle, 23 ± 2°C) and had access to food and water ad libitum. Animal housing care and the application of experimental procedures were in accordance with national and institutional guidelines (Animal Care Committee from CIDEM). The number of mice used, and their suffering was minimized.

Experimental design

First, aged mice with aging-related behavioral damage on spatial working memory, novelty recognition, locomotor activity, and anxiety were selected (Fig. 1) The criteria to classify mice as cognitive impaired or healthy aged animals was based on the mice performance in Ym (spontaneous alternation). The animals were then randomly divided into four experimental groups (n = 16 animals per group): (1) young adults treated with carboxymethylcellulose (CMC); (2) healthy aged treated with CMC; (3) cognitive impaired aged mice treated with CMC; (4) cognitive impaired aged treated mice with JM-20 8 mg/kg. JM-20 was suspended in a 0.05% carboxymethylcellulose (CMC) as vehicle and administered orally (po) at 8 mg/kg for 40 days. The selection of the dose range and administration schedule of the JM-20 to be evaluated was based on previous experience with this compound in dementia models [11]. During the experimental study period, animals from each group were weighed on days − 14, -7, 0, 7, 15, 21, 28, 35 (Fig. 1).

Behavioral assays

Y maze test

Spatial memory and reference memory were examined using the Y maze test (Ym) [15]. The Ym apparatus was constructed of plastic and contained three arms (40 x 20 x 7 cm) with a central platform at 120°. The Ym task consisted of a single session: each mouse was placed on the central platform and allowed to move freely through the maze for 8 min. Alternation was defined as multiple entries into one of the three arms (A, B, or C) using overlapping triplet sets. The percentage of spontaneous alternation was calculated by the following Eq. (2):

$$\:\varvec{\%}\:\varvec{A}=\frac{No.A}{(TE-2)}\times\:100$$

Where:

A:Number of alternations

TE:Total entries

2 and 100:mathematical factor

Novel object recognition test

Novel object recognition test (NOR) is a working memory task and was performed as described previously, with minor modifications [16]. Animals were habituated to the empty rectangular chamber (30 x 30 x 50 cm) for 5 min. NOR was divided into two phases: training and evaluation, of 5 min per trial, separated by an interval of 60 min. In the first trial (training session), animals were exposed to two identical familiar (F) objects. During the second trial (testing session), mice were exposed to one F object, while the second was replaced with a novel (N) object with a different shape and color from the F object. Each mouse was placed between the two objects in the chamber, facing away from the objects. The results were expressed as an index discrimination (ID) between the time objects N and F were explored. The ID was calculated according to the exploration time of each object by the (1) [16]:

$$\:\varvec{I}\varvec{D}=\frac{tN-tF}{tN+tF}$$

Where:

tN:time exploring the new object (in seconds)

tF:time exploring the familiar object (in seconds)

Open field

The open field test (OF) was carried out in a square box 40 x 40 x 40 cm (height x length x width) divided into 16 squares [16]. The squares delimited two spaces: one in the center and another in the and periphery of the box. The animal was placed in the center of the box and its behavior was recorded for 6 min. The following parameters were recorded: total distance traveled by the animals, distance traveled in the center, distance traveled on the periphery, time spent by the animal in the center, time spent on the periphery, and time spent standing still. The data were analyzed in the ImageJ software 1.54f (Bethesda, MD, USA).

Morris Water Maze

Spatial memory was tested using a Morris water maze (MWM) [17] (a circular dark tank with 150 cm diameter, 60 cm height). The water maze was filled with water to a depth of 40 cm. The pool was divided into four equal quadrants (North, South, East, and West) which were used as start points. An escape platform of 11 cm diameter was placed 1 cm below the water's surface at a constant location in the center of the South/West quadrant. The animals were subjected to a daily session of four training trials for five consecutive days in a different escape order for each quadrant. The mice were permitted a maximal time of 90 s to find the hidden platform and allowed to remain on the platform for 30 s before the commencement of the subsequent trial. If the rat failed to locate the hidden platform within 90 s, it was gently placed on the platform and allowed to remain there for a 30 s period. On the 6th day, the platform was removed, and a single departure was made from the North. Evaluating in the same way during 60 s, if the mouse remembered where the platform was. In addition, the number of crossings over the place where the platform was located was determined.

Study of oxidative stress markers

Preparation of brain tissue for enzymatic and redox state assays

Once the behavioral tests had been completed, mice were sacrificed by decapitation; their brains were removed and rapidly dissected to separate the hippocampal regions (HO). Samples were homogenized in sodium phosphate buffer (0.1 mM, pH 7.4: NaCl 0.3M, KCl 2.7 mM, KH₂PO₄ 136.4 mM, Na₂HPO₄ 0.0016M) in a 1:10 ratio (w:v), and centrifuged at 6 000 g for 20 min in an Eppendorf Centrifuge (Germany, 5424R). Aliquots of the homogenates from the brain structures were stored at -80°C until use. The homogenates were used to determine lipid peroxidation malondialdehyde (MDA) mediators, total tissue -SH (T-SH), superoxide dismutase (SOD) and catalase (CAT) activity as previously reported [18]. Protein quantification was made by the Lowry method [19], using bovine serum albumin (BSA) as standard and a POLARstar Omega fluorescence spectrophotometer (BMG Labtech, Offenburg City, Germany) for the absorbance readings.

Assays of acetylcholinesterase enzyme activity in the hippocampus

The activity of the acetylcholinesterase (AChE) in homogenates from the HO was measured spectrophotometrically by the method described by Ellman [20] with slight modifications. The assay was run in a 96-well plate with a final volume of 200 µL. The reaction medium contained 140 µL of Ellman's reagent, 50 µL of homogenate, and 10 µL of a 20mM acetylthiocholine iodide (AcICh) solution and was incubated for 1 minute at 37°C. The absorbance was read at 405 nm for 20 min at 1 min intervals on a POLARstar Omega fluorescence spectrophotometer (Germany). Enzyme activity was calculated and expressed as µmol of (AcICh) hydrolyzed in 1 minute per mg protein.

Statistical analysis

The GraphPad Prism 7.0 program (GraphPad Software Inc., USA) processed and statistically analyzed the results. Data were expressed as mean ± SEM (standard error of the mean) and checked for normality and homoscedasticity. An analysis of variance (ANOVA) was performed in one or two ways, depending on the processing needed, followed by a Tukey multiple comparison test (post hoc) to compare the different experimental groups. A value of p < 0.05 was considered statistically significant. The complete data set obtained from this study is publicly available at [21] https://zenodo.org/records/13222149.

Characterization of the experimental subjects

In the Stage 1 (selection of aged mice with cognitive impairments see Fig. 1) the experimental subjects were categorized in terms of memory performance in three behavioral paradigms (spontaneous alternation in Ym, NOR and the OF performance; Fig. 1). The mice performance in Ym (spontaneous alternation; Fig. 1). This resulted in two aged groups (a healthy aged and a cognitive impaired group). All the young mice had a good performance in the percentage of alternation in the Ym, NOR and OF behavior. From there, aged animals with an alternation equal to or lower than 55% were selected as those with impairment in the spatial working memory

Figure 2A shows the frequency of alternation spontaneous % in the group of aged and young adult mice. From this analysis, the mice that formed the experimental groups were selected: healthy and aged with cognitive impairment. In addition, a comparison was made between these two groups and the young adults. In this case, the mice of the young adult and healthy-aged groups have a % of spontaneous alternation significantly higher than the healthy-aged mice (p < 0.001) (Fig. 2B).

The behavior of young, healthy and cognitively impaired aged mice in the new NOR is depicted in Fig. 2C and 2D. The young and healthy aged mice recognized the new object, but the cognitively impaired animals had no memory and explored the familiar and novel object similarly (Fig. 2C; p < 0.001).

In the OF, healthy young and aged adult mice tended to travel a long distance when compared to aged mice with cognitive impairment (p < 0.05).

Young and healthy cognitive aged mice also stayed for a longer time in the center of the OF. This behavior indicates a higher degree of anxiety for the cognitive impaired aged mice. Which is not necessarily pathological but, rather, constitutes a response to a stressful situation (p < 0.001) (Fig. 2E-F).

Effect of JM-20 on different types of memory in cognitive impaired aged mice

After the initial characterization of aged mice as healthy or cognitively impaired subjects, aged mice with cognitive impairment were randomly separated into two experimental groups: a control group treated with CMC (vehicle) and a group treated with JM-20 8 mg/kg for 40 days. In this second stage of the experimental design, the effect of JM-20 on object recognition, spatial working, and reference memory was evaluated the behavioral tests described in Fig. 1 (stage 2).

In the Ym, the spontaneous alternation performance of the young adult, healthy aged, and cognitively impaired aged mice treated with JM-20 8 mg/kg exhibited a significantly higher % of alternation than the aged group animals with cognitive impairment treated with CMC, (p < 0.05). Taken together with the data presented in Fig. 2B, the data from Fig. 3A indicate that JM-20 rescues the performance impairment in the Ym.

However, healthy-aged and cognitively impaired-aged mice showed impaired memory for N object recognition because they did not discriminate significantly between F and N objects (p > 0.05) (Fig. 3B). In the case of young adult mice and cognitively impaired aged mice treated with JM-20 (8 mg/kg), they explored longer the N object concerning the F object significantly (p < 0.05). An ID with a value close to one means that the animal explored the N object for a longer time with respect to the F one, indicating that it remembers the F object. This shows that recognition memory is not significantly affected in any groups studied (Fig. 3B).

The performance of mice in the second OF exposure is depicted in Fig. 4. Aged animals with cognitive impairment travelled less than to the healthy young and aged adult animals (Fig. 4A). In the OF test, the total distance travelled by the young and aged healthy mice were higher than that of cognitively impaired mice (regardless the treatment with JM-20) (Fig. 4B). In addition, the evaluation of the total motor activity during the 6 min session at 1, 3 and 6 min, indicated that up-to 3 min the activity of all groups were similar, but that in the last 3 min of the 6 min session the activity of cognitively impaired aged mice were lower than the healthy groups (Fig. 4A). o effect of time was observed on ambulatory activity, i.e., the course of the 5 weeks did not modify the exploration of the animals. It can be seen that aged animals tend to decrease their locomotion after this time (Fig. 4B-D).

The effect of JM-20 on the anxiety of the mice was evaluated by measuring the time the animals stayed in the center of the OF arena. The results show that JM-20 administration did not significantly change the time spent in the center of the OF (Fig. 4E). However, a trend towards its increase is observed. It is suggested that animals with higher anxiety will be in the center of the arena for a more extended period.

The MWM is a widely utilized behavioral test to study the spatial reference memory in rodents. The scape latencies to find the platform during the learning sessions are depicted in Fig. 5A. If a mouse did not find the platform within 90 seconds, the animal was removed from the maze and a score of 90 seconds was noted. During the learning phase, there were no statistically significant differences in the escape latency among the experimental groups (p > 0.05). Of note, the latencies of the young and cognitively impaired mice treated with JM-20 mice were numerically lower than the healthy aged and cognitively impaired aged mice. Overall, they tended to learn faster than the healthy aged and damaged cognitively impaired aged mice (Fig. 5A).

During the evaluation phase, the young adult, healthy aged and cognitively impaired aged mice treated with JM-20 mice reached the platform place faster than that of the cognitively impaired group animals (p < 0.05) (Fig. 5B). Accordingly, the young and aged cognitively impaired mice treated with JM-20 mice crossed a greater number of times the place of the platform in the maze, when compared with the cognitively impaired aged mice treated with the vehicle group (p < 0.05) (Fig. 5C). There were no statistically significant differences between f the experimental groups when analyzing the time in the target quadrant (p > 0.05; or the time in the periphery (p > 0.05) Fig. 5D,E).

Body weight

The body weight of aged mice groups was higher than that of young mice during the entire period of the study (p < 0.0001). In fact, the weight of young mice increased as function of time, whereas the adult mice did not exhibit variations in the body weight, indicating that aged mice had already reached their maximum weight (Fig. 6).

Effect of JM-20 on endogenous antioxidant defense systems and lipid peroxidation in hippocampal homogenate

Endogenous antioxidant defense activity was determined by measuring the activity of antioxidant enzymes SOD and CAT, T-SH (Table 1), and MDA (Fig. 7A) levels in hippocampal homogenates. The activity of antioxidant enzymes SOD and CAT had a complex pattern of changes as a function of the treatments. The SOD activity in the young, healthy aged and cognitively impaired aged mice treated with JM-20 groups was lower when compared with the activity in cognitive impaired aged mice treated with vehicle CMC (p < 0.05) (Table 1). T-SH levels did not vary significantly between the evaluated groups (p˃ 0.05). The CAT activity of the young, healthy aged, and the cognitively impaired aged mice treated with JM-20 groups was higher than that of cognitively impaired aged mice treated with CMC group (p < 0.05) (Table 1).

Table 1

Effects of JM-20 on redox parameters in the hippocampal region. Activity of: SOD, CAT, TSH. Experimental groups: young adult, healthy aged, damaged aged with JM-20 8 mg/kg and vehicle (CMC).
Hippocampus (µM)/mg protein	Young adult	Healthy adult	Damaged aged	Damaged aged + JM-20
SOD	17.000 ± 6.673^a	30.688 ± 8.87^a	19.438 ± 5.853^b	26.750 ± 5.298^a
TSH	7.188 ± 1.328^a	7.125 ± 2.062^a	8.000 ± 1.211^a	7.375 ± 2.277^a
CAT	123.813 ± 5.879^b	99.125 ± 6.520^b	166.813 ± 6.794^a	130.438 ± 5.513^b
The data are presented as the means ± SEM (n = 7–10). Different letters: p < 0.05 by one-way ANOVA, followed by Tukey post hoc.

Effect of age and JM-20 on lipid peroxidation and AChE activity in hippocampal homogenate

MDA levels were determined in hippocampal tissue as an indicator of lipid peroxidation (Fig. 7A). No statistically significant differences were obtained for this marker between the mice in the experimental groups evaluated (p˃ 0.05). In relation to the hippocampal AChE activity, healthy-aged mice group exhibited a significant lower AChE activity compared to the other experimental groups (Fig. 7B).

The aging process is linked to a decline in brain functions encompassing cognition, emotion and motor skills. The decline can significantly impact individuals' physical performance and social life [22]. Consequently, brain aging emerges as a risk factor significantly contributing to neurodegeneration [6]. Moreover, aging is a major factor associated with the progression of diseases characterized by the gradual deterioration of memory and cognitive functions, such as Alzheimer's disease. Therefore, the interest in developing treatments specifically targeting memory impairment is imperative.

Building upon the antecedents of JM-20, the current research endeavor was to assess JM-20 impact in a model of cognitive deterioration associated with aging. Notably, the framework of the present study relied on the previous demonstration of the modulatory role of JM-20 in relation to the cholinergic, inflammatory, and energy metabolism in central nervous system of vertebrates. Given these roles, JM-20 is a molecule of interest for ongoing and future research, especially in the context of treating or preventing neurodegenerative diseases.

In the present research, we evaluated the possible protective effect of JM-20 at doses of 8 mg/kg on spatial working, spatial reference, and novelty recognition memory in aged animals with cognitive impairment, selected on the basis of the criterion of 55% alternation in the Ym.

In this way, it was demonstrated that around 50% of the total aged animals presented affectation of the spatial working and reference memory [23, 24]. As well as the memory of novelty recognition and locomotor activity. Indicating the possible affectation of the functionality of the HO, a key brain area for the correct performance of these evaluated tasks [25].

The MWM is very useful for investigating the neurobiological mechanisms involved in learning and spatial memory [24, 26]. An important methodological aspect is that it allowed a comprehensive assessment of the animals' spatial memory, both in the training trials and in the final test, and different measures of their spatial behaviors could be obtained. There is ample evidence of the critical role played by the HO in declarative memory in humans and spatial memory in rodents [27]. Therefore, these behavioral tests made it possible to determine possible damage at the level of these brain structures. Consequently, it allowed the evaluation of the effect of this new therapeutic candidate, which demonstrated its protective power on the memory of JM-20 at a dose of 8 mg/kg.

Although the HO has a relevant role in memory processes and the execution of behavioral tests in animals, such as those performed in this experiment. This work provides new evidence of the therapeutic potential of JM-20 for the treatment of neurodegenerative diseases in which a state of cognitive and memory impairment is manifested. Importantly, JM-20 not only recovered from age-associated memory impairment, but prevented its prolongation.

Reactive oxygen species production is amplified in damaged and impaired mitochondria and implicated in the normal aging process and most known neurodegenerations. Brain mitochondrial function is thought to deteriorate with age and to be an essential and early contributor to aging [6].

To elucidate the mechanism of action through which JM-20 mediates memory protection, considering the established occurrence of oxidative stress during aging and particularly in dementia diseases, including Alzheimer's disease, this investigation focused on assessing the activity of endogenous antioxidant defenses. This evaluation involved measuring the activity of antioxidant enzymes SOD and CAT, as well as the levels of T-SH and MDA, the latter serving as an indicator of lipid peroxidation. Given that JM-20 has demonstrated its ability to protect against oxidative damage in other experimental models, both through enzymatic and non-enzymatic antioxidant defenses [11, 28, 29], these markers of antioxidant status and brain tissue damage were determined in the homogenate of HO tissue in the four experimental groups. Table 1 shows the results of the determination of SOD, CAT, and T-SH, where there is a higher SOD activity in healthy aged and damaged aged treated with JM-20 8 mg/kg groups when compared with the activity of the damaged, aged group (p < 0.05). T-SH levels do not present significant differences between any of the evaluated groups (p˃ 0.05). The CAT activity of the young adult, healthy aged group, and the damaged, aged group treated with JM-20 was lower when compared to the damaged, aged group (p < 0.05). JM-20 maintained the enzymatic activity of SOD, CAT, and T-SH levels in damaged, aged mice with demonstrated cognitive impairment before treatment, with values similar to those of young adult and healthy aged mice, showing a potent protective effect against oxidative events characteristic of aging, which can lead to the development of diseases such as dementia.

The results showed that the healthy old group exhibited a significant decrease in AChE activity in the HO compared to the rest of the experimental groups (Fig. 7B). This first result is essential, as it describes the brain's status regarding cholinergic neurotransmission in aged subjects. In this case, we can corroborate that, in the aged brain of a healthy subject, AChE activity levels are decreased, presumably as an adaptation to the lower number of cholinergic neurons it has concerning the brain of a healthy subject. However, in the cognitively impaired elderly, the enzyme activity levels are shown to be increased, which coherently explains the memory deficit since AChE clears more acetylcholine in a brain with fewer neurons. Consequently, when treatment with JM-20 was administered, we observed that numerically, the activity values remained similar to the old impaired group. However, the subjects treated with JM-20 did not show cognitive impairment. This result leads us to new hypotheses about the possible molecular mechanisms of JM-20 to protect against memory impairment associated with aging or other pathological conditions with memory impairment.

The results provide preclinical pharmacological evidence for the neuroprotective activity of JM-20 as a promising therapeutic candidate for treating memory disorders associated with aging.

Conflicts of interest/Competing interests

The authors declare that they have no conflict of interest.

Ethics Approval

Housing conditions and experimental procedures were carried out following the institutional, national and international guidelines: Animal Care Committee from CIDEM (Havana, Cuba, National Institutes of Health Guide No. 80 − 23, 1978 and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications 8023, 1978)).

Funding statement

This work was partially supported by the Innovation Financial Funding (FONCI-Cuba) project.

Author Contribution

MWG contributed to the designing, performed and planning of the experiments, data analysis and writing of the manuscript. JRS, DHE, YMP performed and designing experiments, analyzed the data and wrote the manuscript. YAI performed experiments and analyzed the data. EGA analyzed the data, wrote and reviewed the manuscript. ASPY: organized the project, financial support and reviewed the manuscript. JBTR analyzed the data and wrote and reviewed the manuscript. LAFF and YNF: designed the research, performed experiments, analyzed the data, organized the project, wrote and reviewed the manuscript.

Data Availability

The complete data set obtained from this study is publicly available at https://zenodo.org/records/13222149. Additionally, the materials utilized in this study, such as any software or protocols, are accessible upon request to the corresponding author.

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Effect of JM-20 on Age-related cognitive impairment in mice

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Compounds and reagents

Experimental animals

Experimental design

Behavioral assays

Y maze test

Novel object recognition test

Open field

Morris Water Maze

Study of oxidative stress markers

Preparation of brain tissue for enzymatic and redox state assays

Assays of acetylcholinesterase enzyme activity in the hippocampus

Statistical analysis

Results

Characterization of the experimental subjects

Effect of JM-20 on different types of memory in cognitive impaired aged mice

Body weight

Effect of JM-20 on endogenous antioxidant defense systems and lipid peroxidation in hippocampal homogenate

Effect of age and JM-20 on lipid peroxidation and AChE activity in hippocampal homogenate

Discussion

Conclusions

Declarations

Conflicts of interest/Competing interests

Funding statement

Author Contribution

Data Availability

References

Additional Declarations

Status:

Version 1