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431 National Journal of Physiology, Pharmacy and Pharmacology 2018 | Vol 8 | Issue 3
RESEARCH ARTICLE
The effect of pramiracetam in the myelination of the hippocampus in the
BALB/c mouse (Mus musculus)
Adrian Cielo S Angeles, Michael B Ples, Rodel Jonathan S Vitor
Department of Biology, College of Science, De La Salle University, 2401 Taft Avenue, Manila, Philippines
Correspondence to: Rodel Jonathan S Vitor, E-mail: rodeljonathan.vitor@gmail.com
Received: August 19, 2017; Accepted: November 09, 2017
ABSTRACT
Background: Pramiracetam is being used, particularly by students, to increase cognitive function and enhance learning and
memory. There are currently sparse data on the effect of pramiracetam, particularly in the brain. One recent study showed
that there is an increased nitric oxide synthase (NOS) in systemically administered pramiracetam. Aims and Objectives: To
determine if there would be a demyelinating effect in the hippocampus that is a sign of neurological damage due to
increasing nitric oxide synthase. Materials and Methods: A total of fifteen mice were given 200 and 600 mg/kg dose of
pramiracetam for qualitative histological comparison of their hippocampal myelination. After administration with food of
the pramiracetam for 75 days, the mice were sacrificed and the brains were then extracted intact, and sagittal sections of the
brain were made and stained with the Klüver-Barrera method. Hippocampi were analyzed using a compound microscope
to observe for demyelination and neuronal degeneration. Results: Based on histological analysis, there were no signs
of demyelination in the hippocampus of the Mus musculus brain. This may indicate that pramiracetam does not cause
demyelination that may be due to increased NO production. Conclusion: Pramiracetam may be safe to take at doses of
200–600 mg/kg without any demyelinating effects in mouse.
KEY WORDS: Demyelination; Hippocampus; Mus musculus; Pramiracetam
INTRODUCTION
Cognitive enhancers are a class of drugs that may elevate
an individual’s cognition particularly their thinking
abilities, which are considered to be meaningful. In a global
study done in 2015, it was determined that at least 1 in 10
healthy individuals has taken at least once stimulants to
improved performance whether at work or while studying,
with participants from New Zealand, the Netherlands, and
Hungary having the most experience in using the drug.[1]
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DOI: 10.5455/njppp.2017.7.0832609112017
National Journal of Physiology, Pharmacy and Pharmacology Online 2018. © 2018 Rodel Jonathan S Vitor, et al. This is an Open Access article distributed under the terms of the Creative
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Nootropics are a class of drugs that act on the central nervous
system and increase cognitive performance, as well as
act as a neuroprotectant, meaning it protects neurons from
injury, disorders, and diseases.[2] Some various drug groups
that are approved by the US Food and Drug Administration
are racetam, cholinergics, acetylcholinesterase inhibitors,
AMPAkines, smart drugs, dopaminergics, nootropic vitamins,
and neurohormones.[1] Although the mechanism of action of
nootropics is still unknown, it is speculated that they improve
cognition be being mildly cholinergic or anti-GABAergic.[3]
Pramiracetam, which belongs to the racetam group, is a
nootropic food supplement that can supposedly increase
cognitive function and enhance learning and memory, as well
as protect neurons in cases of traumatic brain injury.[4] It is a
piracetam derivative, which is licensed in Italy as a cognitive
enhancer, but its development in the United States has been
discontinued.[5] There is evidence that it can increase nitric
National Journal of Physiology, Pharmacy and Pharmacology
Angeles et al. Effects of pramiracetam on hippocampal myelination
National Journal of Physiology, Pharmacy and Pharmacology 432
2018 | Vol 8 | Issue 3
oxide (NO) activity in the cerebral cortex[6] and has shown
to reverse memory defects in rats with cholinergic deficits,
showing possible cholinergic effects.[2] Even though it’s
mode of action is not clear,[5] several potential mechanisms
have been suggested, which includes high-affinity choline
uptake[2] and increasing NO synthase (NOS) activity in the
cerebral cortex in rats.[6] There has been an interest regarding
pramiracetam and other piracetam derivatives in the treatment
of disorders related to Alzheimer’s disease,[7] cognition,
epilepsy, seizures, neurodegenerative diseases, ischemia,
and anxiety disorders.[8] Pramiracetam is considered more
potent than piracetam, pramiracetam’s potency being based
on the dosage requirements compared to piracetam and other
piracetam derivatives. Pramiracetam has shown to improve
cognitive deficits associated with traumatic brain injury[8]
and showed antiamnesic effects on volunteers treated with
scopolamine, an acetylcholine competitive antagonist, that
can cause reversible amnesia.[7] It has been shown that its
main action may be attributed to increasing memory and
learning by synergistically acting with choline, leading to
greater enhancement of cognition.[3] Furthermore, it has
been showed that it also regulates the release of glutamate in
the cortex and hippocampus, suggesting that it has an effect
on N-methyl-D-aspartate (NMDA) receptor function. On the
other hand, although it has low affinity to glutamate receptors,
it causes AMPA receptor stimulation which leads to influx
of calcium in the brain. This leads to the recent reports,
suggesting that it is an acetylcholinergic and glutamatergic
enhancing drug.[1,3] Despite these findings, there have only
been 4 clinical trial publications before 1999 concerning
pramiracetam, possibly because their findings produced
inconclusive results, thus requiring better-controlled studies,
as well as lack of target specificities and exploring possible
long-term risks.[8]
The myelin sheath is a phospholipid layer, formed by
oligodendrocytes in the central nervous system and by
Schwann cells in the peripheral nervous system, that
facilitates synapses and provides structural and biochemical
support for the axons of neurons and speeds up neuronal
conduction.[9] The myelin sheath is segmented and interrupted
at the intervals by the nodes of Ranvier. The segments are
about 0.5–1.0 mm in length. It is the substance that colors the
white matter.[10] Myelination is the process of the formation
of the myelin sheaths around the axons. It begins in the late
fetal development and the forebrains of humans through
early adulthood. It has also been suggested that this process
may continue up to at least the age of 30.[10] Myelination has
been linked to synaptic plasticity, and it has been suggested
that it is more than a developmental process and is related to
cognitive functions, which is shown in developed white matter
structures in children who have increased cognitive function
and motor skills.[11] On the other hand, demyelination is the
effect of damage or injury done to the myelin sheaths that
can lead to slowing down of conduction and transmission of
signals in the nervous system.[12] Demyelination can be caused
by diseases such as multiple sclerosis[13] or prolonged intake
of antipsychotic medications.[14] NO is a free radical that can
perform various roles in the human body, like a signaling
molecule, and moderator of neuronal functions.[15] NO can
be produced in several physiological processes by NOS that
converts L-arginine to NO.[9] NO has been implicated to have
a role in the myelination of the developing[2] brains as well as
being associated with an increase in central nervous system
myelination in rats.[16,17] However, NO has been shown to be
capable of being toxic to oligodendrocytes and can induce
the degeneration of axons that may lead to the process of
demyelination.[17,18]
Since neurons are irreplaceable and given the potential of
pramiracetam to affect NO levels and the relation of NO
to demyelination, it would be a relevant area of inquiry to
explore these relationships further. Knowing the effects,
particularly whether the increased in NO synthesis will lead
to demyelination, will allow a more prudent and scientific
usage of the product. Specifically, the objective of the study
was to determine whether demyelination would result from
the administration of pramiracetam to mice.
MATERIALS AND METHODS
Procurement of Animals
A total of fifteen 8-week-old, weighing around 25–30 g,
female BALB/c mice were obtained from the University
of the Philippines Manila. The sample size was determined
based on the methods of Charan and Kantharia.[19] Animals
were then placed in the Animal House at De La Salle
University and were housed in individual standard sized
cages. Standard commercial rodent food (pellet form) and
drinking water were provided. The cages were lined with
autoclaved paddy husk and cleaned twice a week. Before
the experiment, all mice were acclimatized for 1 week to
adapt to an environment with the temperature of 23°C and
55% humidity at a 12 h light:12 h dark cycle. All succeeding
experiments in animals were approved by the Institutional
Animal Care and Use Committee of De La Salle University
(Reference # 2013-005). The experimental procedures were
performed from September 2014 to April 2015.
Administration of Treatment
The 15 mice were divided into three groups, a control group
and two experimental groups at doses of 200 mg/kg and
600 mg/kg, following the transformed[20] doses from the
study of Corasaniti et al.[6] Pramiracetam was purchased in
powder form and were administered to the mice together
with the food for 75 days. At the end of the study, mouse
was sacrificed through cervical dislocation, and the brain was
harvested for histological analysis.
Angeles et al. Effects of pramiracetam on hippocampal myelination
433 National Journal of Physiology, Pharmacy and Pharmacology 2018 | Vol 8 | Issue 3
Histological Preparation and Analysis
The parenchyma of the brain was extracted intact, which
included the 3 meningeal layers. The extracted brains
were fixed with 10% neutral phosphate-buffered formalin
solution. The tissue samples were then brought to St. Luke’s
Medical Center, Quezon City, for histological processing.
Klüver-Barrera stain was used to demonstrate the presence
of myelin sheaths of the axons in the white matter of the
nervous tissue, as well as demyelination. Histological
analysis was performed with a compound light microscope,
and photodocumentation was done using a Sony DSC-S930
SteadyShot 10.1 Megapixel digital camera.
RESULTS
Histological analysis showed no demyelination in both the
control groups and experimental groups [Figure 1].
The white matter as shown in Figure 1a-c shows no regions
of demyelination, which would be seen as white patches
in the areas stained blue. Any demyelination on the white
matter would be seen as patches of unstained tissue where
the white matter (white arrows) would be. Figure 1d-f
show the pyramidal cells in the hippocampus. The axons
of these pyramidal cells project to the white matter of the
hippocampus and are part of the neuronal circuits present in
the hippocampus, along with the granule cells of the dentate
gyrus as shown in Figure 1g-i, which is densely packed.
We can infer from the results that the white matter of the
200 mg/kg group or the 600 mg/kg group is intact, with the
presence of the intact pyramidal cells, and neuronal damage
of any significance would affect the myelination of the white
matter. This can also be said of the granule cells, as they
project to the pyramidal cells, and account for a large amount
of the neurons in the central nervous system,[21] indicating
that the neural pathways in the hippocampus are intact.
DISCUSSION
The results of the study showed that there is no demyelination
in the brain of the mice that may be due to pramiracetam
administration. This may have a positive impact on the
use of the medication particularly since it is being used for
cognitive enhancement. Since no demyelination was seen, it
is possible that the dosage of 200 mg/kg and 600 mg/kg was
not sufficient to increase levels of NO in the brain. The lack
of demyelination suggests that the pramiracetam doses given
to the 2 experimental groups had no significant effect on the
white matter and consistently show that no demyelination
occurred. While the exact mode of action of pramiracetam is
still not fully understood, the effects of increasing NO activity
may not be sufficient to cause demyelination. Furthermore,
since NO at normal levels may promote myelination,[16] it
may be inferred that pramiracetam, given the clinical trials
that was performed, may actually be protective rather than
damaging to neurons.
Cognitive functions have long been thought to require an
active participation of the hippocampus. The hippocampus, a
three-layered structure in the temporal lobe, has been linked
to long-term memory consolidation and considering that
Figure 1: (a-c) Different groups showing the hippocampus with white matter (white arrows) and dentate gyrus (black arrow). (d-f) Different
groups showing pyramidal cells and glial cells in the hippocampus. (g-i) Different groups showing showing granule cells in the dentate gyrus
d
hi
c
g
b
f
a
e
Angeles et al. Effects of pramiracetam on hippocampal myelination
National Journal of Physiology, Pharmacy and Pharmacology 434
2018 | Vol 8 | Issue 3
the hippocampus is part of the limbic system along with the
amygdala, anterior thalamic nuclei, and the limbic cortex, it can
also be associated with learning, memory consolidation, and
behavior.[9] It has also been shown to exhibit neurogenesis.[22]
Since pramiracetam can affect cognitive functions, it can be
inferred that it can have an effect on the hippocampus. The
acquisition and retention of memories require an active role
of the hippocampus. The cornu ammonis (CA) or horn of
Ammon, part of the hippocampus proper, is differentiated to
four fields such as CA1, CA2, CA3, and CA4. The pyramidal
cells of CA3 and CA4 continue on to the area of the dentate
gyrus, which is also part of the hippocampal formation. The
hippocampus is also part of a major dopaminergic pathway,
called the mesolimbic pathway, which in turn is a part of
the mesolimbic/mesocortical system. The mesolimbic or
mesocortical system is thought to be involved in behavioral
and cognitive responses.[9] Although neurons are amitotic,
there has been research that shows neurogenesis in the dentate
gyrus of the hippocampus of adult mammalian brains, where
they have found neural stem cells.[22]
Pramiracetam, a nootropic racetam derivative drug, has been
used for the purpose of increasing cognition, however, with
a few clinical evidence to support its claim in long-term
memory formation. It has also been assessed in elderly patients
with mild cognitive impairment and arterial hypertension.
Results of the study showed that improvements in attention,
digit span, short-term memory, verbal paired association,
and visual reproduction as well as long-term memory were
present.[23] In another study on patients with head injury and
anoxia, sustained improvement was seen in 18 months of the
treatment even up to 1-month post-withdrawal of the drug.[24]
The cognitive effects of pramiracetam have somehow been
attributed to the accelerated hippocampal acetylcholine
turnover, which increases septal-hippocampal cholinergic
neuronal impulse flow.[25] One study in rats showed that
there is an increasing NO activity in the cerebral cortex of
rats given pramiracetam.[6] It has been attributed that NO in
the hippocampus may have a role in learning and memory
because NMDA receptors are activated during avoidance
tasks in chicks.[26] While the presence of NO is normal
in the central nervous system, high levels can be toxic to
oligodendrocytes and cause axonal degeneration,[17] which is
an integral part of demyelination.[13] Overproduction of NO
has been seen to induce the generation of highly reactive
species such as peroxynitrite and stable nitrosothiols, which
may cause irreversible cell damage.[27] Neurotoxicity has
also been associated with NO in a variety of neurological
disorders including stroke, Parkinson’s disease, and even
HIV dementia.[28] These effects may be attributed to an
increase in different isoforms of NOS all of which may lead
to detrimental effects.[29]
It may thus be inferred from this study that there may be
different effects of administration of pramiracetam in different
test organisms and may thus need further study to verify
whether adverse effects may be seen or not. Furthermore,
without conclusive evidence on the effect of pramiracetam,
careful use of the nootropic is warranted.
CONCLUSION
The results showed no signs of demyelinated nerve fibers
and white matter structures and no significant cell damage.
The dosages given to the mice were based on a study done
on rats given pramiracetam, and the dosage requirements
were adapted to mice. The dosages that were given the rats
showed an increase in NO activity in the rat cerebral cortex,
although in different experimental setups. NO may be a factor
in myelination as well as demyelination, but the administration
of pramiracetam did not cause any such demyelination in this
trial. Pramiracetam at these dosages did not seem to cause
demyelination, and despite its potential to increase NO activity,
the increased levels of NO, if any, did not cause toxicity.
REFERENCES
1. Tripati A, Mathew M, Nayak V, Kurady LB. Cognitive
enhancers-truth vs. hype. Res J Pharm Biol Chem Sci
2016;7:729-41.
2. Mondadori C. In search of the mechanism of action of the
nootropics: New insights and potential clinical implications.
Life Sci 1994;55:2171-8.
3. Patel SJ, Patel KK, Patel MS, Rupak MA, Patel YB, Sanyal AP,
et al. Neuro stimulants cognitive enhancers as nootropics in
multi task hectic schedule. World J Pharm Res 2016;5:570-90.
4. Bambagiotti-Alberti M, Bartolucci G, Bruni B, Coran SA, Di
Vaira M. Diisoprop-yl{2-[2-(2-oxopyrrolidin-1-yl)acetamido]
eth-yl}ammonium hydrogen sulfate. Acta Crystallogr Sect E
Struct Rep Online 2008;64:o1160.
5. Shorvon S. Pyrrolidone derivatives. Lancet 2001;358:1885-92.
6. Corasaniti MT, Paoletti AM, Palma E, Granato T, Navarra M,
Nisticò G, et al. Systemic administration of pramiracetam
increases nitric oxide synthase activity in the cerebral cortex of
the rat. Funct Neurol 1995;10:151-5.
7. Mauri M, Sinforiani E, Reverberi F, Merlo P, Bono G.
Pramiracetam effects on scopolamine-induced amnesia in
healthy volunteers. Arch Gerontol Geriatr 1994;18:133-9.
8. Malykh AG, Sadaie MR. Piracetam and piracetam-like drugs:
From basic science to novel clinical applications to CNS
disorders. Drugs 2010;70:287-312.
9. Rhoades RA, Bell DR. Medical Physiology: Principles for
Clinical Medicine. 4th ed. Baltimore: Lippincott Williams &
Wilkins; 2013.
10. Snell RS. Clinical Neuroanatomy. 7th ed. Baltimore: Lippincott
Williams & Wilkins; 2010.
11. Fields RD. Myelination: An overlooked mechanism of synaptic
plasticity? Neuroscientist 2005;11:528-31.
12. Lublin FD. Clinical features and diagnosis of multiple sclerosis.
Neurol Clin 2005;23:1-15.
13. Olek MK, editor. Multiple Sclerosis: Etiology, Diagnosis and
New Treatment Strategies. Totowa: Humana Press Inc.; 2005.
14. Konopaske GT, Dorph-Petersen KA, Sweet RA, Pierri JN,
Zhang W, Sampson AR, et al. Effect of chronic antipsychotic
Angeles et al. Effects of pramiracetam on hippocampal myelination
435 National Journal of Physiology, Pharmacy and Pharmacology 2018 | Vol 8 | Issue 3
exposure on astrocyte and oligodendrocyte numbers in
macaque monkeys. Biol Psychiatry 2008;63:759-65.
15. Roghani M, Mahboudi F, Saharian MA, Etemadifar M,
Esfahani AN, Nahrevanian H, et al. Concentrations of nitric
oxide metabolites in the serum of iranian multiple sclerosis
patients. J Neurol Sci 2010;294:92-4.
16. Olivier P, Loron G, Fontaine RH, Pansiot J, Dalous J,
Thi HP, et al. Nitric oxide plays a key role in myelination in the
developing brain. J Neuropathol Exp Neurol 2010;69:828-37.
17. Sellebjerg F, Giovannoni G, Hand A, Madsen HO, Jensen CV,
Garred P, et al. Cerebrospinal fluid levels of nitric oxide
metabolites predict response to methylprednisolone treatment
in multiple sclerosis and optic neuritis. J Neuroimmunol
2002;125:198-203.
18. Karpuzoglu E, Ahmed SA. Estrogen regulation of nitric
oxide and inducible nitric oxide synthase (iNOS) in immune
cells: Implications for immunity, autoimmune diseases, and
apoptosis. Nitric Oxide 2006;15:177-86.
19. Charan J, Kantharia ND. How to calculate sample size in
animal studies? J Pharmacol Pharmacother 2013;4:303-6.
20. Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from
animal to human studies revisited. FASEB J 2008;22:659-61.
21. Behesti H, Marino S. Cerebellar granule cells: Insights into
proliferation, differentiation, and role in medulloblastoma
pathogenesis. Int J Biochem Cell Biol 2009;41:435-45.
22. Ross MH, Pawlina W. Histology: A Test and Atlas with
Correlated, Cell and Molecular Biology. 7th ed. Baltimore:
Lippincott Williams & Wilkins; 2015.
23. Bachinskaya N, Demchenko E, Kholin V, Shulkevich A. Use of
pramiracetam in elderly patients with mild cognitive impairment
and arterial hypertension. J Neurol Sci 2013;333:e292-357.
24. McLean A Jr. Cardenas DD, Burgess D, Gamzu E. Placebo-
controlled study of pramiracetam in young males with memory
and cognitive problems resulting from head injury and anoxia.
Brain Inj 1991;5:375-80.
25. Pugsley TA, Shih YH, Coughenour L, Stewart SF. Some
neurochemical properties of pramiracetam (CI-879), a new
cognition-enhancing agent. Drug Res Dev 1983;3:407-20.
26. Rickard NS, Gibbs ME. Hemispheric dissociation of the
involvement of NOS isoforms in memory for discriminated
avoidance in the chick. Learn Mem 2003;10:314-8.
27. Zhao J. Interplay among nitric oxide and reactive oxygen
species: A complex network determining cell survival or death.
Plant Signal Behav 2007;2:544-7.
28. Dawson VL, Dawson TM. Nitric oxide in neurodegeneration.
Prog Brain Res 1998;118:215-29.
29. Liñares D, Taconis M, Maña P, Correcha M, Fordham S,
Staykova M, et al. Neuronal nitric oxide synthase plays a key
role in CNS demyelination. J Neurosci 2006;26:12672-81.
How to cite this article: Angeles ACS, Ples MB, Vitor RJS. The
effect of pramiracetam in the myelination of the hippocampus
in the BALB/c mouse (Mus musculus). Natl J Physiol Pharm
Pharmacol 2018;8(3):431-435.
Source of Support: Nil, Conflict of Interest: None declared.
