The Neuroprotective Effects of Semax in Conditions of MPTP-Induced Lesions of the Brain Dopaminergic System

Abstract

This report describes studies cf the effects of the ACTH(4-10) analog Semax (MEHFPGP) on the behavior of white rats with lesions to the brain dopaminergic system induced by the neurotoxin MPTP. Neurotoxin was given as single i.p. doses of 25 mg/kg. Neurotoxin injections were shown to decrease movement activity and increase anxiety in the animals. Daily intranasal administration of Semax at a dose of 0.2 mg/kg decreased the severity of MPTP-induced behavioral disturbances. The protective activity of Semax in MPTP-induced lesions of the brain dopaminergic system may be associated with both its modulating effect on the dopaminergic system and the neurotrophic action of the peptide.

Full text

ACTH/MSH-like peptides, collectively known as
“melanocortins,” represent a class of endogenous regula-
tory peptides currently subject to active study. This class
of peptides includes adrenocorticotrophic hormone
(ACTH), melanocyte-stimulating hormone (MSH), and
their fragments and synthetic analogs. These compounds
are interesting because peptides of this class have marked
actions on central nervous system (CNS) function. The
first data on the neurotrophic effects of melanocortins
were obtained from studies of their effects on animal
behavior. These studies showed that these peptides act on
learning, motivational processes, and the concentration
and attention abilities of animals. The behavioral effects
of melanocortins are not associated with their hormonal
activity, but result from their direct actions on the CNS
[9]. Structural-functional studies identified the amino acid
sequence for a minimal ACTH fragment retaining its
behavioral activity. The most marked effects on behavior
were seen with the fragment ACTH(4–10) – Met-Glu-His-
Phe-Arg-Trp-Gly, which retains the behavioral effects of
the whole molecule. Tetrapeptide ACTH(4–10) has almost
the same activity [4, 11].
The literature now contains many reports on the effects
of melanocortins on nervous tissue development and regen-
eration processes [12]. Peptides of this class promote neu-
ron survival and the growth of neurites in tissue cultures
[19]. It has also been demonstrated that ACTH, MSH, and
their fragments and analogs have neurotrophic and neuro-
protective influences on the central and peripheral nervous
systems during early ontogenesis and in neuronal damage
in adult animals. Peripheral administration of melanocortins
during the neonatal period accelerates maturation of the
neuromuscular system, affects CNS development, and leads
to long-term changes in animal behavior [18]. In peripheral
nerve lesions in adult animals, administration of
melanocortins accelerates nerve regeneration and muscle
reinnervation, as demonstrated by electrophysiological,
morphological, biochemical, and functional tests [21].
Positive influences of melanocortins have also been noted
in experiments with central nervous system lesions induced
both by transection of various parts of the brain and by
administration of neurotoxins. Peripheral administration of
ACTH fragments and their analogs accelerates functional
recovery after damage to the hippocampus, labyrinthecto-
my, and bilateral lesions and section of the fornix. In addi-
tion, it has been demonstrated that administration of the
ACTH(4–9) analog ORG 2766 has marked protective
actions in lesions to the substantia nigra due to administra-
Neuroscience and Behavioral Physiology, Vol. 34, No. 4, 2004
The Neuroprotective Effects of Semax in Conditions of
MPTP-Induced Lesions of the Brain Dopaminergic System
N. G. Levitskaya, E. A. Sebentsova, L. A. Andreeva,
L. Yu. Alfeeva, A. A. Kamenskii, and N. F. Myasoedov
0097-0549/04/3404-0399
©
2004 Plenum Publishing Corporation
399
Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 88, No. 11, pp. 1369–1377,
November, 2002. Original article submitted June 11, 2002.
This report describes studies of the effects of the ACTH(4–10) analog Semax (MEHFPGP) on the behav-
ior of white rats with lesions to the brain dopaminergic system induced by the neurotoxin MPTP.
Neurotoxin was given as single i.p. doses of 25 mg/kg. Neurotoxin injections were shown to decrease
movement activity and increase anxiety in the animals. Daily intranasal administration of Semax at a dose
of 0.2 mg/kg decreased the severity of MPTP-induced behavioral disturbances. The protective activity of
Semax in MPTP-induced lesions of the brain dopaminergic system may be associated with both its mod-
ulating effect on the dopaminergic system and the neurotrophic action of the peptide.
KEY WORDS: ACTH fragments, Semax, dopaminergic system, MPTP, rats.
Institute of Molecular Genetics, Russian Academy of Sciences,
123182 Moscow, Russia; Faculty of Biology, M. V. Lomonosov
Moscow State University, 119899 Moscow, Russia.

tion of the neurotoxin 6-OHDA. Animals given the peptide
showed accelerated recovery of behavioral, morphological,
and biochemical parameters as compared with controls [6].
Thus, extensive experimental data have now been
accumulated providing evidence of the neurotrophic influ-
ences of melanocortins on the developing and regenerating
nervous system. Use of short ACTH fragments and their
analogs lacking hormone activity allows the endocrine
properties of the hormones to be separated from their neu-
rotrophic effects. Structural-functional studies have demon-
strated that as in the case of the behavioral effects, the neu-
rotrophic activity is due to the N-terminal part of the ACTH
molecule [12]. Immunochemical methods have demonstrat-
ed the existence of an endogenous ACTH(4–10) fragment
in the rat brain during the early neonatal period. In adult
animals, immunoreactivity to ACTH(4–10) has been noted
after lesioning of the nervous system. It has been suggested
that the endogenous ACTH(4–10) fragment is formed in the
brain during the period of nervous system development and
during the regeneration of nerve tissue [7].
A significant disadvantage of natural melanocortins is
their short duration of action. Many investigators have tried
to create highly effective analogs of ACTH fragments by
introducing various modifications of the primary structure
of the molecule. These experiments resulted in the develop-
ment of analogs of natural peptides lacking hormonal
effects but having marked neurotrophic activity and high
protease stability. Examples of such compounds include the
ACTH(4–9) analogs ORG 2766 and HOE-427, as well as
the ACTH(4–10) analog BIM 22015, which have been
shown to have high levels of behavioral, neurotrophic, and
neuroprotective activity in in vivo and in vitro experiments.
These peptides improve learning and memory, accelerate
regeneration of peripheral nerves after transection, and have
neuroprotective actions in various models of pathology [16,
17, 20]. Preliminary clinical studies have supported the data
obtained in animal experiments. However, none of the syn-
thetic analogs listed above has yet been introduced into
clinical practice. The ACTH(4–10) heptapeptide analog
Semax (Met-Glu-His-Phe-Pro-Gly-Pro) has been devel-
oped and studied at the Institute of Molecular Genetics,
Russian Academy of Sciences and the Faculty of Biology,
Moscow State University. Studies of the physiological
activity of this peptide have demonstrated that it improves
memory and attention, has antihypoxic and antihemorrhag-
ic effects, and promotes decreases in the severity of the clin-
ical and neurophysiological manifestations of experimental
ischemic insult. Thus, Semax retains the spectrum of activ-
ity of natural ACTH fragments and the effects are manifest
for prolonged periods [8]. Semax is currently used in med-
icine as a nootropic agent. Clinical studies have shown its
high efficacy in the treatment of cognitive/memory disor-
ders of different origins and in the prophylaxis and treat-
ment of post-anesthesia memory impairments [1].
Administration of Semax has marked positive actions in the
treatment of stroke [3]. Recent studies have established that
the peptide can increase the lifetime of nerve cells in pri-
mary cultures from rat embryo brain [2].
Studies of the neurotrophic effects of Semax continue,
using a variety of experimental models, the aim being to
widen the spectrum of its clinical application. The present
study addresses the effects of Semax on an MPTP-induced
model of parkinsonism. Parkinson’s syndrome arises as a
result of damage to and degeneration of a proportion of the
dopaminergic neurons in the substantia nigra, leading to
decreases in dopamine levels in the striatum [13]. These
lesions can be modeled experimentally by administration of
the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahy-
dropyridine), which specifically damages dopaminergic
neurons in the substantia nigra, inducing the development
of parkinsonian symptoms. Biochemical and structural
changes induced by systemic administration of MPTP are
accompanied by disorders of behavioral reactions and the
development of depressive behavior [14]. The aim of the
present work was to study the efficacy of Semax in condi-
tions of MPTP-induced lesions to the brain dopaminergic
system.
METHODS
Studies were performed on male white mongrel rats
weighing 180–230 g. Animals were kept in standard ani-
mal-house conditions and all experiments were performed
between 11:00 and 18:00. Lesions of the dopaminergic sys-
tem were induced by single injections of MPTP (25 mg/kg,
i.p., in physiological saline). Animals received intranasal
Semax at doses of 0.05 and 0.2 mg/kg 30 min after neuro-
toxin administration. Semax was subsequently given daily
for four days one hour after testing. Each series of experi-
ments involved four groups of animals (18–20 rats per
group); controls received i.p. physiological saline and
intranasal water; the Semax group received i.p. physiologi-
cal saline and intranasal Semax; the MPTP group received
i.p. MPTP and intranasal water; the combined treatment
group received i.p. MPTP and intranasal Semax.
One day after MPTP administration, the animals’
behavior was measured in an open field test (in stress-free
conditions) followed by testing in the burrow box 1 test.
Rats were subsequently tested once daily using the follow-
ing methods: an elevated maze test at two days, burrow box
2 at three days, and in the open field test (stressful version)
at four days.
Open Field Test (OFT). The experimental chamber
was a round arena 80 cm in diameter with a wooden floor
divided by two concentric circles and eight diameters. A
500-W electric lamp was placed 80 cm above the arena,
along with a domestic electric bell and a red 15-W lamp.
For testing, animals were placed in the center of the arena
and horizontal movement activity was monitored visually
Levitskaya, Sebentsova, Andreeva, et al.400

for 2 min, with measurments of excursion length, number of
sector crossings, vertical movement activity (number of
rearings), the number of excursions to the center of the
arena, and grooming (number of washes). Two versions of
the test were performed: the “stress-free” version (in silence
and with the red lamp) and the “stressful” (with the bright
light and bell).
Burrow Box (BB). The experimental apparatus con-
sisted of a square arena with round openings in the floor,
which was divided into squares. Two different experimental
chambers were used – BB1 and BB2. BB1 was a wooden
chamber of size 40 × 40 × 30 cm with 13 openings in the
floor, which was divided into nine squares. BB2 was a
metal chamber of size 47 × 47 × 27 cm with 16 openings,
the floor being divided into 16 squares. The use of two dif-
ferent chambers allowed the animals’ responses to new
experimental situations to be studied in both cases. For test-
ing, rats were placed in the corner of the chamber and the
following measures were recorded visually for 3 min: the
latent period of leaving the start square, horizontal move-
ment activity (excursion length, number of segment cross-
ings), the number of openings investigated, vertical move-
ment activity (number of rearings), and grooming.
Experiments were performed in the quiet with illumination
from the red lamp.
Elevated Cross Maze (ECM). The experimental
chamber was a cross maze (arm length 35 cm, wall height
20 cm). Two opposite arms were dark and closed; the other
two were illuminated and open. The maze was located at a
height of 50 cm from the floor. Rats were placed in the cen-
ter of the maze and the following measures were recorded
for 3 min: the latent period of entering the closed arm, the
total time spent in the light, the number of excursions from
the closed arms, the number of times the animals hung from
the open arms of the maze, and the number of rearings.
Results were analyzed statistically using parametric
(ANOVA) and non-parametric (Wilcoxon–Mann–Whitney
and Fisher) tests run on Statistica. Data were plotted as
mean ± standard error of the mean.
RESULTS
The animals’ behavior was tested one day after MPTP
administration and the first dose of Semax using the open
field and burrow box 1. Two doses of Semax produced no
significant changes in the measures recorded. The MPTP
injection induced significant decreases in horizontal and ver-
tical movement activity in the open field as compared with
controls. Semax (0.05 mg/kg) after MPTP did not alter the
effects of the neurotoxin. Rats given Semax (0.2 mg/kg)
after neurotoxin also showed a significant decrease in excur-
sion length in the open field, though the number of rearings
was insignificantly greater than the level seen in rats given
MPTP only. There were no significant differences in this
parameter either in relation to controls or the levels in rats
given MPTP only (Fig. 1). Studies of the animals’ behavior
in burrow box 1 also revealed decreases in excursion length
and number of rearings after MPTP. Semax (0.05 mg/kg)
after neurotoxin produced no significant changes in the ani-
mals’ behavior as compared to animals given MPTP alone.
However, the number of rearings in this group was slightly
lower than in the MPTP group, though there was no signifi-
cant difference from controls. Horizontal and vertical activ-
The Neuroprotective Effects of Semax in Conditions of MPTP-Induced Lesions 401
Fig. 1. Behavioral parameters in rats in the open field test (stress-free version, one day after MPTP
administration). Vertical axes: A) excursion length, cm; B) number of rearings. 1) Controls; 2) Semax
(0.2 mg/kg); 3) MPTP; 4) MPTP + Semax. *Significant differences from control,
#
significant dif-
ferences from MPTP group.

ity in rats given MPTP and Semax (0.2 mg/kg) reached the
control level and was significantly different from the level in
rats given neurotoxin only (Fig. 2).
Animals were tested in the elevated cross maze two
days after MPTP (by this time, the rats had received two
injections of Semax). Animals given Semax (both doses)
showed no significant changes in behavioral parameters in
the maze as compared with controls. Animals given MPTP
showed significant decreases in the total time spent in the
illuminated part of the maze and the number of times they
hung from the open arms compared with controls. In addi-
tion, the number of excursions from the closed arms showed
no significant difference from control (p = 0.99). Semax
(0.05 mg/kg) after MPTP produced no increase in the num-
ber of hangings from the arms of the maze compared with
animals given neurotoxin alone. In animals given MPTP
and Semax (0.2 mg/kg), parameters such as the total time
spent in the light, the number of excursions from the closed
arms, and hangings from the maze arms were significantly
higher than values in the group given MPTP and were no
different from those in controls (Fig. 3).
Animals were tested in burrow box 2 three days after
MPTP. Semax (both doses) produced so significant changes
in parameters. Rats given neurotoxin showed significant
Levitskaya, Sebentsova, Andreeva, et al.402
Fig. 2. Behavioral parameters in animals in the burrow box 1 test (one day after administration of MPTP).
For further details see caption to Fig. 1.
Fig. 3. Behavioral parameters in animals in the elevated cross maze test (two days after MPTP). Vertical axes: A) time spent in open arms, sec;
B) number of excursions from closed arms; C) number of hangings from the maze arms. For further details see caption to Fig. 1.

reductions in horizontal and vertical movement activity, as
well as increases in the latent period of leaving the start
square, as compared with controls. Semax (0.05 mg/kg)
produced no significant increase in excursion length or the
number of rearings compared to the group given MPTP.
Rats given Semax (0.2 mg/kg) after neurotoxin
showed a significant increase in excursion length and a
decrease in the latent period compared with animals given
MPTP only. In addition, the number of rearings in this
group was not significantly different from that in rats given
neurotoxin (p = 0.07) (Fig. 4).
Animals were tested in the stressful version of the
open field test four days after MPTP. Chronic administra-
tion of Semax (both doses) had no effect on the animals’
behavior in this test. Rats given MPTP showed significant
decreases in excursion length and the number of excursions
to the center of the field as compared with controls. Semax
(0.05 mg/kg) had no effect on behavioral changes induced
by MPTP. Rats given Semax (0.2 mg/kg) also showed a sig-
nificant decrease in excursion length compared with con-
trols, like the MPTP group, though the number of excur-
sions to the center of the field made by this group was
The Neuroprotective Effects of Semax in Conditions of MPTP-Induced Lesions 403
Fig. 4. Behavioral parameters of animals in the burrow box 2 test (three days after MPTP). Vertical axes: A) latent period of leaving the
start square, sec; B) excursion length, cm; C) number of rearings. For further details see caption to Fig. 1.
Fig. 5. Behavioral parameters of animals in the open field test (stressful version) four days after MPTP. Vertical
axes: A) excursion length, cm; B) number of excursions to the center. For further details see caption to Fig. 1.

significantly greater than in rats given neurotoxin only and
was no different from the level in controls (Fig. 5).
DISCUSSION
Studies of the animals’ behavior in the open field and
burrow box tests showed that MPTP led to decreases in hor-
izontal and vertical movement activity as compared with
controls. Neurotoxin-induced changes in the rats’ behav-
ioral parameters in the elevated cross maze test provided
evidence for an increase in the level of anxiety. The
decrease in the number of excursions to the center of the
arena in the stressful version of the open field test seen in
rats given MPTP may also be associated with increases in
anxiety. These data lead to the conclusion that single doses
of MPTP (25 mg/kg) induce decreases in movement and
orientational-investigative activity in rats and increase the
level of anxiety. The behavioral changes persisted for at
least four days after neurotoxin administration.
Semax (0.05 mg/kg) given after MPTP in some cases
decreased the extent of behavioral disorders induced by
neurotoxin. However, none of the experiments showed sig-
nificant differences between this group of animals and those
given MPTP only.
Semax (0.2 mg/kg) after MPTP-induced lesioning of the
dopaminergic system to a significant extent normalized the
animals’ behavior. Rats of this group were characterized by
higher levels of motor and orientational activity and decreased
levels of anxiety as compared with animals given MPTP only.
Thus, daily intranasal administration of Semax
(0.2 mg/kg) promoted functional recovery in animals with
lesions to the dopaminergic system of the brain, having
protective effects in the MPTP-induced model of parkin-
sonism. Semax (0.05 mg/kg) was virtually ineffective in
this model.
ACTH-like peptides are known to have modulating
effects on the brain dopaminergic system [10] – they
increase dopamine concentrations in various brain struc-
tures, activating the synthesis and release of the transmitter
in response to external stimuli. Previous studies have
demonstrated that administration of Semax weakens behav-
ioral abnormalities in animals induced by the dopamine
receptor blocker haloperidol, which may be associated with
increases in transmitter release [5]. The modulating influ-
ence of Semax on the brain dopaminergic system may
underlie the neuroprotective effects of the peptide in MPTP-
induced lesions. Increases in the synthesis and release of
dopamine by undamaged neurons may lead to increases in
the transmitter concentration in the striatum to the level typ-
ical of the asymptomatic stage of parkinsonism.
Semax has also been shown to increase neuron sur-
vival in primary cultures from the basal nuclei of the fore-
brain and to increase the expression of the genes encoding
the neurotrophic factors NGF and BDNF in glial cell cul-
tures [15]. Thus, the effects seen here may be associated
with the neurotrophic activity of Semax, due to weakening
of damage and restoration of the function of reversibly
damaged nigrostriatal dopamine-synthesizing neurons.
Thus, the neuroprotective effects of Semax in
MPTP-induced lesions to the brain dopaminergic system
may be based on both compensatory processes needed for
functional recovery and increases in neuron survival.
This study was supported by the Russian Fund for
Basic Research (Grant No. 01-04-48767).
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