Main Research Findings

1) Regulation of the AMPA receptor through treatment with aniracetam resulting in an improvement in neurological deficits and a reduction of infarct percentage due to the compound’s neuroprotective and anti-inflammatory properties.

2) Treatment with aniracetam in vitro was shown to decrease the level of apoptotic cells as well as improve cellular oxidative stress by reducing the amount of reactive oxygen species present.

 

Selected Data

1) The research team of Sharma et al assessed the effects of treatment with aniracetam on the activity of AMPA receptors in the acute and subacute phase of neurological damage post-stroke. For the purpose of this study 14-16 week old male Wistar rats weighing between 240 and 280 grams. The test subjects housed 4 rats per cage and maintained under standard laboratory conditions with ad libitum access to water and dry pellets. Animals were allowed to acclimatize for one week before being randomly assigned to different experimental treatment groups. It is important to note that only male rats were used in this study in order to prevent the neuroprotective effects of estrogen from skewing the collected data. The animals were anesthetized with 5% isoflurane anesthesia while cerebral ischemia was induced using an intraluminal technique meant to occlude the middle cerebral artery that was removed after 90 minutes [1].

There were several objective measures being monitored by the research team during this study. First, the effects of the AMPA receptor, perampanel, on various neurobehavioral parameters after a 1.5 mg/kg dose was administered 10-15 minutes post reperfusion. The second objective examined the effects of perampanel on various neurobehavioral parameters after a 1.5 mg/kg dose was administered 10-15 minutes post reperfusion, for 3 days. The third objective examined the effects of perampanel on various neurobehavioral parameters after a 1.5 mg/kg dose was administered 10-15 minutes post reperfusion, for 5 days. Objective 4 introduced aniracetam and assessed the effects of a 50 mg/kg dose of AMPA receptor agonist when administered 3 days after reperfusion, while objective 5 assessed the effects of a 50 mg/kg dose of aniracetam administered 5 days after reperfusion. For objective 6 the research team identified the time at which perampanel and aniracetam were at its most effective in terms of the reduction of infarct percentage and motor incoordination. These time points were used for secondary experimental purposes [1].

The next portion of the study included a neurobehavioral assessment based on a neurological deficit score, a rota rod test, and a grip strength test. After a middle cerebral artery stroke was induced, the neurological deficits experienced by the test subjects was scored on a 5 point scale; a score of 0 indicated no neurological deficits; a score of 1 includes a failure to fully extend the right forepaw; a score of 2 is indicated by circling to the right; a score of 3 is defined by falling to the right; and a score of 4 includes the lack of spontaneous walking and an overall decreased levels of consciousness [1].

On the other hand, the rotarod test examined the motor coordination of the rats both before and after the induction of a middle cerebral artery stroke. The animals were allowed a single training session with the rotating rota rod apparatus; the rotarod moved at a constant speed of 8 mph and the training session continued until the animals could remain on the rota rod for 60 consecutive seconds. Following training the animals received a baseline trial where the rota rod speed increased from 4 mph to 40 mph over a period of 5 minutes, the same examination occurred on day 7 post occlusion [1].

The last neurobehavioral exam was the grip strength test performed both before and after the reperfusion injury occurred. The test included an apparatus elevated 40 cm from the surface and consisted of a 50 cm long strong pulled taut between two vertical support beams. The test subject was placed at a midway point between the two support surfaces and evaluated on a 6 point scale; a score of 0 indicates that the animal fell of the string; a score of 1 includes hanging onto the string by two forepaws; a score of 2 is characterized by an attempt to climb on back on to the string; a score of 3 is defined as hanging onto the string by two forepaws plus one or both hindpaws; a score of 4 includes hanging onto the string with all paws plus the tail wrapped around the string; and a score of 5 means that the animal was able to escape the apparatus by traveling to one of the support systems [1].

2) The research team of Gabryel et al examined the capability of aniracetam to protect cultured astrocytes against ischemic injury. Following the acquisition of the necessary chemicals and materials, astrocytes were isolated from 1-day old Wistar rats and cultured according to standard procedure. The cells were plated according to whether they were destined for bioluminescent study, fluorescent study, caspase-3 activity analysis, RT-PCR analysis, or Hoechst 33342 staining [2]. The astrocyte samples were cultured in a medium composed of 20% FBS which was replaced after 4 days with a medium of 10% FBS; after 1 week the entire volume of culture medium was changed twice a week while the cultures shaken overnight to remove any contaminating non-astroglial cells. In order to identify astrocytes the cultures were immunohistochemically stained for GFAP analysis; the researchers noted that all experiments were performed on 21-day cultures [2].

The experimental procedures began by culturing normoxic astrocytes in DMEM medium with 10% FBS and 5.5 mM glucose, followed by placing the cultures in DMEM with 3% O2/5% CO2/92% N2 for 24 hours in order to mimic in vivo ischemia. Aniracetam was dissolved in DMSO at an initial concentration of 10 mM; the cell cultures were then treated with varying doses of the compound ranging from 1, 10 and 100 uM for 24 hours in normoxia and an additional 24 hours of simulated ischemia. The kinase inhibitor, PD98059, was added to the culture medium at a concentration of 50 mM, followed by the addition of wortmannin to the culture at a concentration of 0.1 mM [2].

Next, apoptosis was assessed by staining the cell nuclei by Hoechst 3342 to visualize fragmented and condensed DNA. The astrocytes were washed with PBS and cultured for 10 minutes with 4% paraformaldehyde; after a second washing with PBS the samples were then dehydrated in 70% ethanol first, and then dehydrated again in absolute ethanol. The cells were stained with Hoechst 33342 and washed with PBS for a final time, allowing for cell nuclei analysis with fluorescence imaging to take place using the MiraCal Pro III workstation and an inverted microscope Eclipse TE200. 6 randomly selected areas from each cell culture plate was examined in order to determine the number of apoptotic nuclei [2]. Each region contained approximately 200 cells and the results were based on a calculation of the percentage of apoptotic cells relative to the total number of cells found in a region.

Concentrations of ATP and creatine phosphate were examined through the use of high-specific firefly luciferin-luciferase bioluminescence assay. The conversion of PCr to ATP was catalyzed by the addition of creatine kinase followed by the measurement of ATP concentrations by bioluminescence quantification. These procedures include disrupting the cells found in 100 ul samples and adding them to 5% TCA solution with 2 mM EDTA followed by rapid vortexing and incubation. The samples were then diluted 20-fold with a pH 7.75 Tris-acetate buffer. 10 uL of the ATP concentration was then added to each cellular plate as an internal standard and allowed the researchers to count the total amount of ATP present [2].

The researchers determined the enzymatic activity of the caspase-3 compound through the use of a caspase-3 colorimetric assay that utilizes the release of fluorochrome p-nitroaniline (p-NA) in combination with a caspase-3 specific peptide substrate. These parameters were chosen by the research team considering that peptide cleavage through active caspase-3 has been shown to release chromophore p-NA that can be quantified using a 405 nM colorimetric plate reader. The underlying mechanism behind quantification included lysing cultured astrocytes and centrifuging the cell extracts to eliminate any debris [2]. 20 uL of the cell extracts were then incubated with the substrate and the level of caspase-3 activity was calculated as a proportion of the color reaction intensity to control values.

Additionally, the conversion of 2’,7’-DCF-DA into fluorescent DC mediated by the presence of reactive oxygen species was examined in order to determine the levels of cellular oxidative stress. The cultured astrocytes were exposed to 100 uM DCF-DA and incubated for 50 minutes followed by washing three times with HBSS while a fluoroscan microplate reader was used to quantify DCF fluorescence. The dye used for this portion of the study was excited at 485 nm and the emission was filtered using a barrier set at 538 nm; the research team determined the production of reactive oxygen species as a percentage of the control cells.

Finally, RT-PCR analysis was completed in order to determine the expression of c-fos and c-jun genes in cultured rat astrocytes samples after being exposed to ischemic conditions in 24 hours and treated with varying doses of aniracetam. Total RNA was isolated from astrocytes and cultured on 100 mm culture cell plates. The RNA was precipitated and purified followed by dissolving in sterile nuclease-free water and stored for further use. Isolated RNA was quantified and the reverse transcription reaction was performed using 100 ng of total RNA [2].

Amplification of gene expression was carried out for 30 cycles for c-fos, c-jun, and beta-actin primers; these primers used in the PCR analysis corresponded to a specific DNA sequence for each of genes. RT and PCR were performed via Termoblock thermocycler and 10 uL of the amplified PCR products were stained by ethidium-bromide in 1% TAE buffer and analyzed by densitometry. All experiments were completed three times and results were quantified using Image Pro Plus software, followed by normalization in accordance with the expression of the beta-actin gene [2].

 

Discussion

1) It was initially confirmed that occlusion of the middle cerebral artery resulted in significant increases in neurological deficits and decreased scores on the grip strength and rota rod tests. The results of the first three objectives assessing the efficacy of common AMPA receptor agonist, perampanel, found that when the compound was administered 3 and 5 days after induction of middle cerebral artery stroke, neurological deficits were significantly reduced. While administration at both of these time periods decreased the infarct percentage, 5 days post occlusion was found to experience maximum recovery. The same results were seen in the rota rod and grip strength tests where maximum recovery was seen on day 5 post occlusion. The primary difference between the rota rod and grip strength tests and the neurological deficit scoring was that the AMPA receptor agonist was administered on day 1 post occlusion as well as days 3 and 5 [1].

Figure 1: Changes in A) neurological deficit score, B) grip strength score, C) time spent on the rotating rota rod apparatus in response to treatment with parampanel after middle cerebral artery stroke.

In terms of objective 4 and 5, it was initially confirmed that occlusion of the middle cerebral artery resulted in an increase in neurological deficit scores as well as decreased scores on the rota rod and grip strength testing. The results of this portion of the study found that treatment with aniracetam at both 2 and 5 days after ischemic reperfusion injury had the potential to elicit maximum protection against neurological deficits. In terms of grip strength testing, treatment with aniracetam administered at both 3 and 5 days after ischemic reperfusion injury improved grip strength scores dramatically. However, it is important to note that administration of the compound on day 5 elicited stronger effects than when administered on day 3. Finally, the results of the rota rod test found that when aniracetam was administered the animals spent a considerably longer amount of time within the rotating rota rod apparatus. Improvement in performance and motor coordination was found to be most significant when aniracetam was administered 5 days post ischemic reperfusion injury [1].

Figure 2: Changes in A) neurological deficit score, B) grip strength score, C) time spent on the rotating rota rod apparatus in response to treatment with aniracetam after middle cerebral artery stroke.

Finally, objective 6 examined the effects of sequential treatment with perampanel and aniracetam on neurological deficits, muscle strength evaluated by the grip strength test, and motor coordination evaluated by the rota rod test. Following induction of middle cerebral artery ischemia significant neurological impairments were identified that were ultimately resolved following sequential treatment with perampanel and aniracetam. Similar results were seen for the grip strength test and the rotating rota rod test, indicating an improvement in muscle strength and motor coordination, respectively, after sequential treatment with perampanel and aniracetam [1].

Figure 3: Changes in A) neurological deficit score, B) grip strength score, C) time spent on the rotating rota rod apparatus in response to sequential treatment with parampanel and aniracetam after middle cerebral artery stroke.

2) When examining the results of Hoechst 33342 staining in order to determine the effects of aniracetam on apoptosis. After the cell cultures were exposed to 24 hours of simulated ischemic conditions, the number of apoptotic nuclei was significantly increased. When treating the cultures with varying doses of aniracetam, the number of apoptotic nuclei was markedly reduced with the 100 uM dose of the nootropic eliciting the most potent effects against ischemia-induced apoptosis. The research team also investigated the role of PI 3-kinase and MAP kinase/MEK in the antiapoptotic mechanism of aniracetam. That being said, 0.1 uM of a PI 3-kinase inhibitor, wortmannin, and 50 uM of MEK inhibitor, PD98059, were used to help the researchers further define the mechanism behind cell death prevention [2].

Figure 4: Changes in the percentage of apoptotic cells measured by Hoechst 33342 in response to treatment with 1, 10, or 100 uM of aniracetam

The cell cultures that were treated with wortmannin and PD98059 during exposure to ischemic conditions resulted in 86 +/- 7.3% and 38.7 +/- 6% of the cells died, respectively. When administering 100 uM of the aniracetam in combination with wortmannin or PD98059 decreased the number of apoptotic nuclei to 3.9 + 1.3% and 11.8 + 2.9%, respectively. These findings allowed the research team to conclude that aniracetam has a significant influence on MEK and PI 3-kinase pathways [2]. A 100 uM dose of the nootropic also had the potential to prevent the process of apoptosis when exposed to ischemic conditions.

Following exposure of the astrocyte cell cultures to simulated ischemic conditions, the cells were treated with doses of aniracetam ranging from 1, 10, and 100 uM in order for the researcher to examine the influence of the compound on level of creatine phosphate and ATP. Treatment with aniracetam was found to significantly increase ATP concentrations, however, there were no remarkable effects elicits on the levels of creatine phosphate in the samples [2]. Additionally when observing the influence of the nootropic on caspase-3 activity, 1 um of aniracetam did not elicit any significant effects on the enzymatic activity while both the 10 and 100 uM doses were found to significantly decrease activity of caspase-3 in samples exposed to ischemic conditions [2].

Figure 5: Changes in intracellular ATP and PCr in response to treatment with 1, 10, or 100 uM of aniracetam.

Figure 6: Changes in caspase-3 enzymatic activity in response to treatment with 1, 10, or 100 uM or aniracetam.

The researchers also evaluated the effects of aniracetam on cellular oxidative stress marked by the presence of reactive oxygen species. Aniracetam was found to cause a dramatic reduction of DCF fluorescence in the astrocyte samples, indicating a marked decrease in the production of reactive oxygen species. The astrocytes were also subjected to ischemic conditions in combination with the kinase inhibitor PD98059, resulting in an increase in the production of reactive oxygen species. This effect was attenuated when 100 uM of aniracetam was used to treat the astrocyte cell cultures [2].

Figure 7: Changes in the production of reactive oxygen species in response to treatment with 1, 10, or 100 uM or aniracetam.

Similar results were seen when examining the effects of the compound of the expression of the c-fos and c-jun genes. 24 hours of exposure to simulated ischemic conditions led to an increase in the expression of both genes and treatment with 10 and 100 uM of aniracetam for 24 hours was found to significantly decrease gene expression. It is important to note that the 1 uM dose of the nootropic did not elicit any significant effects on gene expression [2].

Figure 8: Changes in expression of the c-fos and c-jun genes in response to treatment with 1, 10, or 100 uM or aniracetam.

 

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] Sharma H, Reeta KH, Sharma U, Suri V, Singh S. AMPA receptor modulation through sequential treatment with perampanel and aniracetam mitigates post-stroke damage in an experimental model of ischemic stroke. Naunyn Schmiedebergs Arch Pharmacol. 2023 Dec;396(12):3529-3545. doi: 10.1007/s00210-023-02544-z. Epub 2023 May 25. PMID: 37231168.

[2] Gabryel B, Adamczyk J, Huzarska M, Pudełko A, Trzeciak HI. Aniracetam attenuates apoptosis of astrocytes subjected to simulated ischemia in vitro. Neurotoxicology. 2002 Sep;23(3):385-95. doi: 10.1016/s0161-813x(02)00084-0. PMID: 12387365.

 

Aniracetam is a fat-soluble molecule belonging to the racetam family. Aniracetam is closely related to the popular nootropic compound, piracetam, but is often considered more effective. The differences between the two compounds stem from its structural components; aniracetam replaces piracetam’s amine group with a methylated phenyl group in order to increase fat solubility. Similar to many other racetam compounds, aniracetam is initially metabolized through the hepatic system, however, evidence shows that it is also efficiently metabolized through the gut, even in a fasted state. The compound is best known for its effects on neurotransmission as well as its ability to modulate excitatory AMPA receptors. That being said, aniracetam is being examined for its ability to treat depression and anxiety
as well as memory impairments.

Animal-based studies have examined the effects of aniracetam acting as an AMPA receptor modulator. Aniracetam works as an effective modulator of AMPA receptors through its mechanism of binding to a non-active site on the receptor. The compound then works to allosterically modify the binding site, leading to a reduced rate of desensitization when excitatory stimuli, such as glutamate, interact with the receptor. Regulation of the AMPA receptors is important to note as they drive most of the excitatory transmission in the brain. This results in accelerated amino acid transmission and improved memory retrieval skills.

Aniracetam has also shown promise in regulating the actions of kainate receptors, compounds that are closely related to the action of glutaminergic transmission and AMPA receptor activity. Kainate receptors are assembled from 5 different glutamate-related units, GluK1-5. Researchers Lowry et. Al examined how removing one of these subunits affects memory in mice. Initially, GluK4 knockout mice were subjected to a water memory maze and exhibited drastic memory acquisition and recall impairments.

These mice also showed signs of hyperactivity and impaired pre-pulse inhibition, two hallmark symptoms of schizophrenia and bipolar disorder. When comparing the GluK4 knockout mice to a wild-type control group, vast differences in memory were noticed and led researchers to believe GluK4 may also play a role in regulating psychiatric disorders. Results reported that the water maze the mice were subjected to is heavily dependent on the functioning of the hippocampus. Proper hippocampal functioning is also linked to schizophrenia and memory loss.

Evidence also suggests that both schizophrenia and bipolar disorder are related to glutaminergic transmission in the brain, specifically the activity of GluK4. This allowed researchers to conclude that neuropsychiatric disorders and neurodegeneration, such as memory loss, could potentially be caused by the improper functioning of Glu4K. However, more extensive research must be done to fully understand the transmission pathways in the brain, as well as how aniracetam could remedy these issues by acting as a positive modulator for kainate and AMPA receptors (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3595388/).

Aniracetam has also shown potential in increasing levels of cholinergic and dopaminergic transmission. Dopamine and acetylcholine (ACh) are two neurotransmitters that are essential to the treatment of anxiety and depression as well as the improvement of memory retention and learning. Animal-based studies have found that aniracetam is capable of potentiating signaling of nicotinic receptors through protein interactions. Additionally, when given 50 mg/kg doses of aniracetam, rats exhibited decreased turnover rates of dopamine resulting in improved mood and memory.

Due to its ability to regulate AMPA and kainate receptors, administration of aniracetam improves neuroprotection and memory loss. A notable study conducted by Cumin et. Al induced memory and learning impairments via cholinergic antagonists, ischemia, and electroconvulsive shock. Results of this study found that doses of approximately 1.5 g can reverse the impairments and protect against new trauma. Furthermore, two different studies reported that the regulation AMPA receptor can lead to an increase in brain derived neurotrophic factor (BDNF), a compound widely known for its ability to improve neural plasticity. The stimulation protocol followed resulted in a sustained increase in BDNF levels for 5 days.

Additional studies noted aniracetam’s ability to affect neurotransmitters such as dopamine, serotonin, and ACh. When administering doses of aniracetam varying from 10-100 mg/kg, researchers noted that anxiety was drastically reduced due to the numerous interactions between the compound and the neurotransmission systems. A notable study conducted by Nakamura et. Al emphasized how the enhancement of dopaminergic signaling via aniracetam was effective at having an antidepressant effect in rat test subjects at doses of 100 mg/kg. It’s important to note that the metabolite of aniracetam, haloperidol and mecamylamine elicited these same effects (https://examine.com/supplements/aniracetam/research/#jlBkq25-overview-1).

The nootropics sold by Umbrella Labs are sold for laboratory research only. The description above is not medical advice and is for informative purposes only.

Aniracetam is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.