Main Research Findings

1) Piracetam has been shown to have neuroprotective functioning on electrophysiological properties of cortical neurons found within peri-infarcted tissue following focal cortical injury.

2) In vitro and in vivo experimental studies have found that concentrations of Piracetam have the potential to improve mitochondrial dysfunction associated with age-related decline and oxidative stress.

 

Selected Data

1) Based on previously acquired data of cortical brain mapping studies and research focused on the enhancement of functioning following ischemic brain injury, the research team of Xerri et al, examined the changes that occur in somatotropic cortical regions adjacent to an area of infarct; the effects of administration of Piracetam on the lesion and the lesion-induced reorganization in the site of injury; the influence of enriching or impoverished environments on the remodeling of the cortical regions adjacent to the lesion; and the effect of chronic treatment with Piracetam on the function organization of remaining intact regions in the somatosensory cortex.

52 male, 3-month old Long-Evans rats that weighed from 300-350 grams were used in this portion of the study for the purpose of electrophysiological brain mapping. The test subjects were weaned 30 days after birth and each litter of rats were housed for 2 months in Plexiglass cages. During this time the research team recorded their neuronal activity within the cortical area of forepaw representation in order to find each subjects’ baseline activity. The recording was followed by the induction of a small neurovascular lesion in the same area of forepaw representation [2]. Twenty of the test subjects were used to record any changings in brain mapping over the first 12 hours after the induction of an ischemic lesion. The remaining 32 rats were used to record any change in brain mapping that took place over the first 3 weeks after the induction of an ischemic lesion.

For the first group of rats the spread of the lesion was evaluated by recording neuronal activity from hours 3 to 4 (T2), 7 to 8 (T3), and 11 to 12 (T4), post-lesion. Piracetam or a placebo compound was administered to the test subjects at 1 hour, 5 hours, and 9 hours after induction of the ischemic lesion occurred. The second group of rats was administered Piracetam or a placebo compound 1 hour after the lesion was induced and once the boundaries of the ischemic lesion were determined the test subjects were returned to either an enriched or impoverished housing environment [2].

All test subjects in the enriched environment lived with 10 other male rats in a spacious cage containing mobile and immobile objects of different shapes and textures to promote tactile exploration and development. The rats living in an impoverished environment were housed alone in small cages without any stimulating objects [2]. 8 rats in both the enriched environment received Piracetam, 8 rats in the impoverished environment received Piracetam, 8 rats in the enriched environment received a placebo treatment, and 8 rats in the impoverished environment received a placebo treatment. Administration of the test subjects’ respective treatments took place twice daily for 3 weeks.

The research team continued to measure vascular landmarks on the surface of the lesioned cortex in order to evaluate the post-lesion cortical tissue loss. Loss of cortical tissue was estimated by demarcating an area of infarction and a polygon of tissue surrounding it. After 3 weeks a new polygon of cortical tissue with an infarct core at the center was determined and tissue loss was estimated by measuring the difference between the two polygons. This measurement took place for each test subject and allowed for the research team to calculate the relative decrease in the infarcted area present in the portion of the cortex representing the forepaw, glabrous and hairy skin surfaces, and nail movement [2].

2) Previous research has found that treatment with Piracetam in various animal models has the potential to enhance the fluidity of aged synaptosomal and mitochondrial membranes while also improving impaired brain functioning related to age-related cognitive decline, as well as hypoxia, trauma injury, and general neuronal degeneration. Current research conducted by the team of Keil et al tested the effects of Piracetam on mitochondrial dysfunction following oxidative stress applied to both PC12 cell and dissociated brain cells of mice treated with Piracetam [3].

PC12 cells were initially cultured in DMEM and supplemented with 10% heat-inactivated fetal calf serum, 5% heat-inactivated horse serum, 50 ug ml streptomycin, 50 U ml penicillin, and 400 ug ml G418. Culturing of the cells with these compounds took place in a humidified incubator containing 5% CO2 and kept at 37℃. The PC12 cells were treated in three different ways. They were first treated with SNP for 24 hours with Piracetam included into the mixture 30 minutes after the exposure to SNP. This treatment was utilized to test the neuroprotective effect of Piracetam after oxidative stress was applied. The second treatment included pretreating the cells with SNP for 30 minutes then exchanging the medium in order to administer treatment Piracetam for 23 hours. Finally, the PC12 cells were pretreated with Piracetam for 6 hours while the mitochondrial membrane potential was recorded, followed by the addition of complex inhibitors.

In order to measure the mitochondrial membrane potential of the PC12 cells, the samples were plated the day prior in a 24-well plate followed by the measurement of the membrane potential with the fluorescence dye R123, as the transmembrane distribution of the dye is reliant of the membrane potential of the mitochondria. The dye was then added to a cell culture medium for 15 minutes followed by washing of the PC12 cells with a balanced salt solution and the fluorescence was examined with the use of a specialized reader [3].

To collect dissociated brain cells from mice treated with the nootropic, 2-3 months old and 22-24 months old female mice were obtained from the Naval Medical Research Institute for the portion of this study. The animals were maintained in standard conditions and provided food and water ad libitum. They were then treated with doses of Piracetam varying from 100, 250, or 500 mg/kg once daily for 2 weeks, while control animals were treated on the same schedule but administered 0.9% NaCl instead [3].

24 hours after the test subject’s last feeding and treatment administration, the mice were euthanized and the brains were dissected followed by mincing of the tissue into 2 ml of a medium containing NaCl 128 mmol/l, KCl 5.4 mmol/l, Na2HPO4 0.17 mmol/l, K2PO4 0.22 mmol/l, glucose 5.5 mmol/l, and sucrose 58.4 mmol/l. This solution was further titrated with a nylon mesh and pasteur pipette followed by filtration of the resulting suspension through a fresh nylon mesh in order for it to be washed with a medium and centrifuged for a final time [3]. The cells were resuspended in 6 ml of DMEM and plated accordingly in order for optimal measurement of the mitochondrial membrane potential. To determine the effects of Piracetam on recovery of the dissociated brain cells after the induction of oxidative stress, the samples were treated with SNP for 6 hours with Piracetam added 1 hour after initial exposure to SNP.

Additionally, caspase 9 activity was measured by plating PC12 cells and pretreating them for 22 hours with Piracetam followed by the addition of SNP to the plates for 2 hours. The cells were harvested and centrifuged allowing for the cell-containing pellet to be washed with PBS and lysed in a buffer containing 10 mM HEPES, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM EDTA, 1 mM dithiothreitol, 1 ug ml pepstatin A, 1 ug ml leupeptin, 0.1% CHAPS, and pH 7.4). The resulting lysate was centrifuged again and the remaining supernatant was used to perform the caspase assay. Activity of caspase 9 was determined by observing the cleavage and production of the colorimetric substrate Ac-LEHD-pNA over the course of 30 minutes [3].

 

Discussion

1) The results of the study found that after the first hour (T1) following the induction of an ischemic lesion and prior to treatment with Piracetam or a placebo, the test subjects exhibited similar electrophysiological activity in the injured cortical regions. Between T1 and T2 the lesion was found to increase in size, however, this increase was similar between both experimental groups. Between T2 and T4, the placebo group showed a significant expansion of the lesion from 0.42 +/- 0.34 mm to 0.91 +/- 0.52 mm, while the lesion in the test subjects’ administered Piracetam remained relatively stable, from 0.31 +/- 0.26 mm to 0.24 +/- 0.36 mm [2[. When setting a baseline area of forepaw representation prior to induction of the lesion, the researchers found that there was no significant difference in the baseline level between any of the test subjects regardless of their experimental conditions.

Following induction of the ischemic lesion, expansion of the lesion was shown to be similar to the previously listed findings. That being said, the average loss of cutaneous representational area was calculated 12 hours after the lesion and reported significantly smaller measurements of the infarct area in the rats treated with Piriacetam. When the research team compared the recorded brain mapping, the results showed that there was an increase in remodeling of forepaw representations within the spared territories. However, it is important to note that general reorganization was common in each experimental group regardless of their treatment [2].

Figure 1: Effects of Piracetam on remodeling of the forepaw representation of the somatosensory cortex.

In order to examine how much cortical tissue was lost after 3 weeks following the induction of an ischemic lesion, the research team evaluated the test subjects in group II and any changes that occurred in the forepaw reference areas related to glabrous skin, hairy skin, and nail movement. The injured area of the cortex was assessed through electrophysiological recording 2 hours post-lesion; this area was expressed as a percentage of the whole cutaneous zone relating to the forepaw map and its different sectors. The recordings found that the sectors of the forepaw reference map serving the glabrous skin and nail movements were more extensively injured than the sectors serving hairy skin. The sectors serving glabrous skin and nail movements were found more medially in the forepaw reference map while the sectors serving hairy skin were represented along the lateral margin of the map [2].

After 3 weeks, examination of the individual forepaw maps found that representational changes occurred in territories of the somatotopic representation of the forepaw that were spared from damage. Furthermore, there was an overall decrease and fragmentation of the regions of the cortex serving skin surfaces surrounding the area of ischemic infarct [2].

The spared area of the cutaneous maps for the group of test subjects administered a placebo compound and housed in an impoverished environment measured at 0.52 +/- 0.30 mm; the spared area of the cutaneous maps for the group of test subjects administered Piracetam and housed in an impoverished environment measured at 0.70 +/- 0.30 mm; the spared area of the cutaneous maps for the group of test subjects administered a placebo compound and housed in an enriched environment measured at 0.70 +/- 0.40 mm; and the spared area of the cutaneous maps for the group of test subjects administered Piracetam and housed in an enriched environment measured at 1.29 +/- 0.41 mm [2].

Figure 2: Post-lesion decrease in total area of forepaw cutaneous representation in response to administration of Piracetam or a placebo compound and housing in an impoverished or enriched environment

2) When examining the effects of Piracetam on the consequences of induced oxidative stress, the research team of Keil et al utilized PC12 cells and cell samples derived from the brains of test subjects treated with Piracetam. The results of the R123 fluorescence staining revealed that when the nootropic was added to the cell culture 30 minutes after the onset of SNP exposure there was an improvement in the mitochondrial membrane potential, as well as the rate of ATP production. Piracetam was also shown to enhance ATP production and mitochondrial membrane potential recovery when Piracetam was exchanged with the medium directly following exposure to SNP [3].

Figure 3: Changes in mitochondrial membrane potential and levels of ATP when A) PC12 cells were incubated with SNP for 24 hours and Piracetam was introduced to the medium 30 minutes into the incubation period; and when B) PC12 cells were incubated with SNP for 30 minutes followed by the addition of Piracetam for 23 hours.

The research team also assessed changes in the activity of caspase 9 after exposure of the PC12 cell samples to SNP to induce oxidative stress, followed by exposure to Piracetam. Caspase 9 activity is important to take note of considering that it leads to the activation of executor caspase 3 and the extrinsic apoptotic pathway, and ultimately results in apoptotic cell death. Caspase 9 was found to activate after exposure to SNP, reaching maximum activity levels 2 hours after induction of oxidative stress. However, when the cell samples were incubated with Piracetam for 22 hours after SNP exposure caspase 9 activity was shown to be significantly reduced [3].

Figure 4: Changes in activity levels of caspase 9 following SNP exposure in control groups, groups exposed only to SNP, and groups exposed to SNP and treated with Piracetam

After following the procedure to prep dissociated brain cells in vitro from mice treated with Piracetam, the data collected by the research team reported that there was no significant difference in the potential of the mitochondrial membrane between the in vitro dissociated brain cells and the in vitro untreated control cells and the cells treated with Piracetam [3]. However, it is important to note that treatment with Piracetam was shown to have protective effects against mitochondrial dysfunction induced by exposure to SNP. This result was specifically seen in the aged mice of 22-24 months of age but not in young mice of 2-3 months of age.

When assessing the brain cells of both aged mice to young mice, older mice experienced significant changes in mitochondrial membrane potential following induction of oxidative stress. In comparison to the previously mentioned in vitro data, Piracetam was shown to have a more dramatic effect in aged mice compared to younger mice. Results of this portion of the study found that treating the test subjects with the nootropic for 14 days had the potential to normalize basal levels of the mitochondrial membrane potential in the brain cells collected from aged mice [3].

Figure 5: Changes in mitochondrial membrane potential A) after exposure to SNP and treatment with Piracetam in dissociated brain cells of young mice; B) after exposure to SNP and treatment with Piracetam in dissociated brain cells of aged mice. C) Stabilization of basal levels of mitochondrial membrane potential following treatment with piracetam. D) Protection against mitochondrial damage following NO exposure and treatment with Piracetam.

In addition to exposing the cell samples to SNP and NO, the cells were also exposed to H2O2 in order to induce a reduction in mitochondrial membrane potential in both young and aged mice treated with Piracetam. Similar to the previous findings of the study, treatment with the nootropic was shown to only elicit protective effects against mitochondrial dysfunction in aged rats [3]. Consistent with exposure to NO, Piracetam was found to normalize basal levels of mitochondrial membrane potential while also protecting against mitochondrial dysfunction induced by H2O2. The research team noted that the first beneficial effects of Piracetam were seen at the lower concentrations included in the experiment, starting at 250 mg/kg [3].

Figure 6: A) Basal levels of mitochondrial membrane potential in aged mice were stabilized following treatment with Piracetam. B) Mitochondrial damage induced by exposure to H2O2 is ameliorated by treatment with Piracetam in a concentration dependent manner. C) In a concentration dependent manner, Piracetam was found to protect against mitochondrial damage induced by exposure to SNP.

 

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] Wheble PC, Sena ES, Macleod MR. A systematic review and meta-analysis of the efficacy of piracetam and piracetam-like compounds in experimental stroke. Cerebrovasc Dis. 2008;25(1-2):5-11. doi: 10.1159/000111493. Epub 2007 Nov 22. PMID: 18033952.

[2] Xerri C, Zennou-Azougui Y, Coq JO. Neuroprotective effects on somatotopic maps resulting from piracetam treatment and environmental enrichment after focal cortical injury. ILAR J. 2003;44(2):110-24. doi: 10.1093/ilar.44.2.110. PMID: 12652006.

[3] Keil U, Scherping I, Hauptmann S, Schuessel K, Eckert A, Müller WE. Piracetam improves mitochondrial dysfunction following oxidative stress. Br J Pharmacol. 2006 Jan;147(2):199-208. doi: 10.1038/sj.bjp.0706459. PMID: 16284628; PMCID: PMC1615864.