Main Research Findings

1) Administration of Emoxypine succinate has been shown to regulate behavioral and molecular responses to iron overload-induced neuroinflammation by targeting the CDK5/GSK3-beta and NLRP3 inflammatory pathways.

2) Following trauma to the brain, treatment with Emoxypine succinate led to strengthened compensatory-restorative process, accelerated recovery of impaired functioning, and decreases in the rate of retrograde degeneration.

 

Selected Data

1) Excessive iron accumulation in the brain is associated with the development of neurodegenerative conditions. The research team of Parab et al examined the effects of Emoxypine succinate as a treatment for neurodegeneration by reducing the negative effects of iron imbalances. For the purpose of this study 90, 6-8 month old, wild type zebrafish were obtained from the Central Institute of Fisheries Education. The subjects were allowed a 2 week acclimatization period to a 30 liter housing tank with continuous or diffused aeration provided through an aeration pump air stone attachment. The tanks were maintained at a constant temperature while the water quality was assessed ensuring a pH between 6.8 and 7.5, ammonia concentrations under 0.02 mg/L, nitrate levels below 50 mg/L, and nitrite levels below 0.1 mg/L. The zebrafish were also kept on a 14 hour light and 10 hour dark cycle with three daily feeding times [2].

After the 2 week acclimatization period, the zebrafish were randomly assigned to 6 groups each including 15 test subjects. The zebrafish were placed in individual 2 liter test tanks either filled with water, to act as a control group, or water containing 1.5 mg/L of ferrous sulfate to induce iron overload neurodegeneration. After 28 in the test tanks the zebrafish were then administered an 8 mg/L dose of deferiprone or Emoxypine succinate in 4 mg/L, 8 mg/L, and 12 mg/L. Both Emoxypine Succinate and deferiprone were administered to the test subjects by dissolving the compound in a 500 ml beaker of water and placing the zebrafish in it for 30 minutes everyday for 14 days. After 14 days of treatment the zebrafish underwent behavioral testing including the Y maze test and the novel tank test [2].

The Y maze utilized a glass tank apparatus that included three arms each measuring 25 cm in length, 15 cm in height, and 8 cm in width with white floors to allow for video analysis and a contrast in color between the fish and the rest of the maze. Black adhesive plastic sheets were applied to the outer maze walls to obscure the outside while colored paper cues in various geometric shapes were applied to the inner walls of the tank. The research team noted that they excluded the color blue from this experiment due to recent studies suggesting zebrafish have an aversion to the color.

The three apparatus arms were the starting arm, the open arm, and the novel arm. The fish were all initially placed in the starting arm at the beginning of both the training and testing trials. The open arm was accessible throughout both the training and testing trials for the zebrafish to explore through the experiment. The novel arm was closed during the initial training sessions but was opened by the research for the purpose of the testing trials. The training trial was performed first, followed by the testing trial that took place 1 hour later; the first training trial lasted 5 minutes and the zebrafish were allowed to explore both the start arm and the open arm. During the second testing trial the novel arm was opened and the zebrafish were allowed to explore all three arms over an experimental period of 5 minutes. The trials were recorded and analyzed in order to determine the total distance each zebrafish traveled and how much time was spent in each arm of the apparatus [2].

The locomotor activity and anxiety levels of the test subjects were assessed through the novel tank test that took place 24 hours after the Y maze test was completed. The novel tank used was in a trapezoid shape measuring 30 cm in length, 12 cm in width, and 15 cm in height with partitioning separating the tank into upper, middle, and lower sections. The zebrafish were placed into this new tank and their activity was recorded over an experimental period of 5 minutes and analyzed to determine the total distance traveled, the average velocity, the latency to entency the top section of the tank, and the time each zebrafish spent in the top and bottom zones of the tank. Immediately after the behavioral tests were completed the zebrafish were euthanized by immersing them for 10 minutes into cold water maintained at a temperature of 0-4 degrees Celsius. The brains of the test subjects were then dissected, weighed, and cleaned in an isotonic saline solution prior to preservation for further analysis.

The brain was homogenized using a 10-fold volume of 0.1 M phosphate buffer solution to assess malondialdehyde levels and perform a catalase test on the resulting homogenates. Additionally, the activity levels of Acetylcholinesterase (AChE) were assessed using a colorimetric AChE assay kit that uses 5,5’-dithiobis-(2-nitrobenzoic acid)) to identify and quantify the production of AChE hydrolytic byproduct, thiocholine. It was also mentioned that an Iron colorimetric Assay Kit was used to quantify the iron content in the brains’ of the zebrafish by isolating ferric ions from within the brain homogenate. Finally, ELISA assay kits were used to evaluate levels of IL-1-beta, TNF-alpha, CDK-5,GSK-3-beta, and NLRP3 in the brain homogenates [2].

2) In order to assess the effects of Emoxypine succinate on conditioned reflexes after a traumatic brain injury, researcher T.A. Mering utilized Wistar rats and completed experimentation through a food-related motor method. Conditioned reflexes to actual and trace stimuli were developed in the rats training them to count time intervals and the duration of a conditioned signal, as well as determine temporospatial differentiation and orientation in space.

The study began by placing the rats in a chamber containing a reinforcement platform for the rats to climb onto when presented with the conditioned stimulus. A sound was then used with a strict temporal stereotype in order to create a conditioned reflex to the sound. The conditioned signal duration lasted for 5-10 seconds and there was a 60 second break between each delivery of a stimulus. After the fixation of the conditioned reflex to sound, the sound was gradually taken away with each trial until the rats transitioned to operate based on time rather than the sound. The conditioned reflex to time was considered acquired and correct once there was no more than one excursion between signals in a 60 seconds period of time [3].

In order to acquire temporospatial differentiation the test subjects were placed at the start of a T or Y shaped maze. A sound was presented for 10 seconds and the rats received food in the right-hand sector of the maze, followed by sound presentation for 3 seconds and food in the left-hand sector of the maze. The signals were given randomly while the researcher team recorded data on the number of correct and incorrect responses, as well as null reactions where the subjects refused to decide a sector and responses including an instant correction of an error [3].

These experiments took place over a period of 12-15 months; the animals were then assigned to three different groups. Group 1 animals were used to investigate the acquisition and current state of the conditioned reflex over prolonged periods of time without surgical intervention. Group 2 animals were used to assess conditioned reflexes after lesioning of the hippocampus. Group 3 animals were used to assess the state of the conditioned reflex after lesioning the hippocampus and being treated with Emoxypine succinate. Lesioning to the hippocampus occurred by injecting the excitatory amino acid, quinolinic acid into the region via microsyringe. If assigned to group 3, the rats were administered Emoxypine succinate twice daily by mixing a water soluble version of the nootropic into the drinking water. There were a total of three one-month treatment courses with a 30 day interval between each course [3].

 

Discussion

1) Results of the Y maze test were based on the number of entries into each arm of the apparatus and the total distance traveled by each zebrafish. The positive control group was shown to spend a longer amount of time in the novel arm and a longer overall distance traveled compared to the negative control group. The zebrafish included in the negative control group experienced compromised performance shown through decreased time spent in the novel arm and a shorter total distance traveled. This indicates that these subjects had increased anxiety, impaired spatial memory, and reduced exploratory tendencies and locomotor activity. Treatment with all three doses of Emoxypine succinate were found to produce positive outcomes in comparison to the negative control group; these subjects experienced a significant increase in the amount of time spent in the novel arm and the total distance traveled. The findings suggest that the nootropic helps attenuate spatial memory deficits while reducing anxiety and increasing exploratory and locomotor activity [2].

Figure 2: B) The total distance traveled by each of the experimental treatment groups and C) the number of entries in the open arm, starting arm, and novel arm by each of the experimental treatment groups.

The results of the novel tank test were determined based on the total distance traveled, the average velocity, latency to enter the top section of the tank, and the amount of time spent in both the top and bottom sections of the tank. The positive control group exhibited longer exploratory times of the upper section of the tank and less time spent in the lower section, an increased total distance traveled and average velocity, and a shorter latency period to enter the top section in comparison to the negative control group. These positive outcomes were also seen in the zebrafish treated with all three doses of Emoxypine succinate, indicating heightened curiosity and reduced anxiety. Additionally, it was noted that the group of subjects treated with 12 mg/L of the nootropic experienced the highest improvement in spatial memory amongst the experimental treatment groups [2].

Figure 3: B) The total distance traveled by each of the experimental treatment groups; C) the average velocity recorded in each of the treatment groups; D) the latency of each of the treatment groups to enter the top section of the tank; E) the amount of time spent in the top section of the tank by each treatment groups; and F) The amount of time spent in the bottom section of the tank by each of the treatment groups.

Oxidative stress in the zebrafish was assessed by tracking the levels of malondialdehyde (MDA), catalase, superoxide dismutase, and glutathione. MDA was significantly higher in the negative control group, indicating oxidative damage and elevated lipid peroxidation. There was also diminished superoxide dismutase and catalase activity, suggesting impaired antioxidant defense mechanisms, as well as reduced glutathione levels, indicative of poor cellular redox balancing. Treating the zebrafish with Emoxypine succinate led to a dose-dependent reduction in oxidative stress. In all three experimental treatment groups there was a significant decrease in MDA levels, especially in the 8 mg/L and 12 mg/L group, suggesting decreased oxidative damage and lipid peroxidation. Activity levels of catalase, superoxide dismutase, and glutathione were all shown to significantly increase, indicating improvements in antioxidant defense mechanisms, the catalysis of the reactive oxygen species, and cellular redox balance [2].

Figure 4: A) Levels of MDA; B) catalase; C) superoxide dismutase; and D) glutathione (GSH) in the experimental treatment groups.

The negative control group exhibited a significant increase in the activity levels of acetylcholinesterase, suggesting that dysfunction-induced neurodegeneration is associated with impaired cholinergic functioning. However, cholinergic functioning was found to improve following treatment with Emoxypine succinate exhibited by a decrease in AChE activity. These effects were found to be dose-dependent with the 12 mg/L dose of Emoxypine succinate eliciting maximum reduction in the activity of the enzyme. These findings allowed the researchers to infer that the nootropic therapeutically addresses neurodegeneration characterized by cholinergic dysfunction through the restoration of cholinergic neurotransmission [2].

Figure 5: Levels of AChE activity in the different experimental groups.

Additional outcome measures assessed included the accumulation of iron in the brain and levels of the following inflammatory markers: IL-1-beta, TNF-alpha, CDK-5, GSK-3-beta, and NLRP3. In comparison to the negative control group, all three of the experimental groups treated with Emoxypine succinate were found to significantly decrease the susceptibility of iron accumulation, suggesting the potential of the nootropic to regulate brain iron homeostasis and attenuate iron-overload related to neurodegeneration. In regards to the upregulation of inflammatory markers, administration of Emoxypine succinate resulted in significant decreases in IL-1-beta, TNF-alpha, CDK-5, GSK-3-beta, and NLRP3 in comparison to the negative control group. These results indicate that treatment with the nootropic has potential anti-inflammatory effects, as well as the ability to regulate key signaling pathways in order to alleviate neuroinflammation and abnormal signaling related to iron dysregulation in the brain [2].

Figure 6: Levels of iron in the brain in the different experimental treatment groups.

Figure 7: Levels of A) IL-1-beta; B) TNF-alpha; C) CDK-5; D) GSK-3-beta; and E) NLRP3 in the different experimental treatment groups.

2) The results of this study found that in the control group of Wistar rats there were 10-30 different sound combinations developed during the experiment and that the number of correct responses reached 90-100%. These levels of correct responses were typically maintained in these animals throughout the study period. Analysis of the data found that acquisition of a conditioned reflex to a sound and the conditioned reflex to time were based on the proportion of correct responses and the accuracy of counting the time interval. Ultimately, the proportion of correct responses and the time counting accuracy is a measurement of the displacement of intersignal responses to the end of the interval [3].

During the initial acquisition period of the conditioned reflex, there were several intersignal reactions distributed throughout the entire interstimulus interval. After the conditioned response was correctly performed 100 times in response to the sound, intersignal reactions were rarely elicited by the rats. That being said, as the sound started to be omitted the amount of correct conditioned reflex responses ranged from 70-100% and maintained stable at an average of 95%. Both intact rats and rats with lesions to the hippocampal region experienced difficulty with the acquisition of temporospatial differentiation. In rats with hippocampal lesions the average number for correct responses was 65%, the average number of incorrect responses was 11%, and the average number of refusals was 24%. In 60% of the rats with a hippocampal lesion treated with Emoxypine succinate, acquisition of the conditioned reflex to time was significantly improved in comparison to lesions subjects receiving no treatment [3].

It is important to mention that acquisition of the conditioned reflex to time took place at the same rate as the lesioned rats not treated with the nootropic. However, it was noted that the animals treated with Emoxypine succinate experienced a continuous learning process. The rats treated with the nootropic only had the correct response 22.8% of the time prior to the fixation of the conditioned reflex, but after acquisition of the reflex over a period of 2 months, the amount of correct responses increased to 72%. Treatment with Emoxypine succinate was also shown to be associated with improved autonomic and emotional-motivational reactions, as well as an increase in lifetime. It was found that 10 of the 11 animals with hippocampal lesions and treated with the nootropic were still alive by the presentation of the 750th sound combination. This was in comparison to the rats that had a hippocampal region and received no treatment where 4 out of the 13 animals did not survive until sound combination 750 [3].

Additionally, monitoring of the hippocampal cells found that the injection of quinolinic acid primarily induced damage to cells in fields CA1, CA3, CA4, and sometimes to the dentate fascia. Generally, the posterior regions of the hippocampus experienced more damage after the injection than the anterior regions. Overall, based on the collected data the research team was able to conclude administration of Emoxypine succinate after trauma to the hippocampus provided a constant acquisition of a conditioned reflex to time and enhanced compensatory-restorative processes related to impaired functions [3].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Shchulkin AV, Mylnikov PY, Chernykh IV, Esenina AS, Yakusheva EN. Pharmacokinetics of Succinate in Rats after Intravenous Administration of Mexidol. Bull Exp Biol Med. 2023 May;175(1):54-58. doi: 10.1007/s10517-023-05810-5. Epub 2023 Jun 20. PMID: 37338763.

[2] S. Bagwe Parab, G. Kaur, Emoxypine succinate modulates behavioral and molecular responses in zebrafish model of iron Overload-Induced neuroinflammation via CDK5/GSK3- β and NLRP3 inflammasome pathway, Brain Research (2024), doi: https://doi.org/10.1016/j.brainres.2024.149236

[3] Mering TA. The action of mexidol on the state of conditioned reflex activity after traumatic brain lesions. Neurosci Behav Physiol. 2003;33(2):133-138. doi:10.1023/a:1021713629090

 

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