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Sleep is an essential physiological process that underlies crucial cognitive functions as well as emotional reactivity. Thus, Sleep Deprivation (SD) may exert various deleterious effects. In this study, we aimed to examine the adverse behavioral and hormonal effects of SD and a potential treatment with Plant-derived nanoparticle treatment - cocc 30c.The study was a 4-arm trial with randomization and double-blinding of verum and placebo treatments. SD was induced by using the Multiple Platform Method for 48 hours. The effects of SD were evaluated behaviorally (Pre-pulse inhibition, startle response and rotor-rod) at baseline as well as at 6, 12, 24 hours, and 14 days post deprivation. cocc 30c treatment was administrated Per Os every three hours starting immediately after baseline tests and for a period of 24 hours. On day 14, blood samples were taken and serum levels of corticosterone, testosterone, serotonin and leptin were tested. We found that cocc 30c improved Pre-pulse inhibition 12 and 24 hours post deprivation, likewise, cocc 30c improved motor learning. On day 14 SD led to increased startle response that was ameliorated by cocc 30c. Likewise, SD led to increased levels of corticosterone and serotonin while decreasing testosterone and leptin. Interestingly, cocc 30c treatment has moderated these hormonal alterations. We conclude that the treatment with cocc 30c recovers both short-term behavioral and the long-term hormonal modulations following SD.
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The Rappaport Faculty of Medicine, Behavioural Neuroscience Lab,
Technion – Israel Institute of Technology, Haifa, Israel
Emek Medical Center, Afula, Israel
The International Institute for Homeopathy Research, Hod
Hasharon, Israel
Yezrel Valley College, Afula, Israel
Abstract—Sleep is an essential physiological process that
underlies crucial cognitive functions as well as emotional
reactivity. Thus, sleep deprivation (SD) may exert various
deleterious effects. In this study, we aimed to examine
the adverse behavioral and hormonal effects of SD and
a potential treatment with Plant-derived nanoparticle
treatment – cocc 30c. The study was a 4-arm trial with
randomization and double-blinding of verum and placebo
treatments. SD was induced by using the Multiple Platform
Method for 48 h. The effects of SD were evaluated behavior-
ally (pre-pulse inhibition (PPI), startle response and rotor-
rod) at baseline as well as at 6, 12, 24 h, and 14 days post
deprivation. cocc 30c treatment was administrated Per Os
every three hours starting immediately after baseline tests
and for a period of 24 h. On day 14, blood samples were
taken and serum levels of corticosterone, testosterone,
serotonin and leptin were tested. We found that cocc 30c
improved PPI 12 and 24 h post deprivation, likewise, cocc
30c improved motor learning. On day 14 SD led to increased
startle response that was ameliorated by cocc 30c. Likewise,
SD led to increased levels of corticosterone and serotonin
while decreasing testosterone and leptin. Interestingly,
cocc 30c treatment has moderated these hormonal altera-
tions. We conclude that the treatment with cocc 30c
recovers both short-term behavioral and the long-term hor-
monal modulations following SD. Ó2013 IBRO. Published
by Elsevier Ltd. All rights reserved.
Key words: attention, nanoparticles, cocc 30c, motor
learning, sleep deprivation, hormones.
Sleep deprivation (SD)
In humans, sleep is an essential physiological process
which, when deprived, may exert deleterious effects. In
the literature, SD is divided into three categories: long-
term total SD (>45 h); short-term total SD (645 h); and
partial SD (sleep restriction to <7 h/24 h) (Durmer and
Dinges, 2005). Considering the above, acute total SD of
24–48 h was previously shown to impair the
performance in both attention (Blagrove et al., 1995;
Bocca and Denise, 2006; Kendall et al., 2006) and
working memory (Wimmer et al., 1992; Smith et al.,
Positron Emission Tomography (PET) scans
confirmed that 24 h of SD decreases glucose
metabolism and synaptic activity in the prefrontal cortex,
an area involved in attention processes, as well as in
dorsal and ventral thalami (Thomas et al., 2000; Kato
et al., 2000). Kato et al. (2000) showed that SD led to
an increase in blood pressure and a decrease in muscle
sympathetic nerve activity. Furthermore, Van leeuwen
et al. (2009) found that SD increases the risk of
cardiovascular diseases by augmenting pro-
inflammatory responses. Other studies have also
showed results of severe symptoms such as irritability,
fatigue, hallucinations, and delusions (Orzel-
Gryglewska, 2010). Overall, SD in humans has been
found to impair attention (Alhola and Polo-Kantola,
2007; McCoy and Strecker, 2011), cognitive functions,
and behavioral performance (Curcio et al., 2006). While
SD’s deteriorating behavioral effects are suggested to
be normally recovered (Schwierin et al., 1999;Faraut
et al., 2012), the duration needed for this recovery was
found to be dependent on the deprivation paradigm.
Specifically, longer SD requires a longer normal
recovery period (McCoy and Strecker, 2011).
Treatment of SD commonly involves psycho-stimulants
such as caffeine, which may restore attention. However
they are not effective when evaluating cognitive tasks,
decision-making or motor activities (Killgore et al., 2012).
Evidence of the efficiency of homeopathic treatment
with cocc 30c given to patients suffering from SD has
been accumulated in our clinic (unpublished data).
Patients who received cocc 30c remedy reported an
improvement in their ability to sleep, reduced anxiety/
0306-4522/13 $36.00 Ó2013 IBRO. Published by Elsevier Ltd. All rights reserved.
*Corresponding author. Address: Behavioural Neuroscience Lab, The
Rappaport Faculty of Medicine, Gutwirth Building, Technion – Israel
Institute of Technology, Haifa 32000, Israel. Tel: +972-50-6503361;
fax: +972-4-6495231.
E-mail addresses:,
(A. Avital).
These authors contributed equally to this work.
Abbreviations: 5-HT, 5-hydroxytryptamine; ANOVA, analysis of
variance; D1R, dopamine receptor1; ITI, Inter-Trial-Interval; MPM,
Multiple Platform Method; PPI, pre-pulse inhibition; SD, sleep
Neuroscience 253 (2013) 1–8
irritability as well as improvement in their cognitive
capabilities. These results were based upon clinical
observations and patients’ reports, thus requiring
systematic validation.
Homeopathy treatment, although partially disputable,
is prevalent both in clinics and research (Lucertini et al.,
2007; Mishra et al., 2011). Accumulating data indicate
detectable effects of different types of remedies (Bell
et al., 2011a), and biological effect of homeopathic
treatment on physiological measures of sleep (Bell
et al., 2011b). Furthermore, in 2012, the Swiss
Government published a health technology assessment
of homeopathy that found strong evidence supporting
homeopathic treatment at least in some medical
treatments (Bornho
¨ft and Matthiessen, 2012).
Specifically, already in 1927 Boericke depict that plant
source material Cocculus has a potential for reversing
the effects of sleep loss (Boericke, 1927).
Finally, new basic science data suggest that the
source materials of the medicine do survive and persist
in nanoparticulate form across homeopathically
prepared potencies, including cocc 30c, as a function of
the unique manufacturing processes (Chikramane et al.,
2010, 2012). Biological effects may be mediated in part
by endogenous adaptive responses (Bell and Koithan,
2012). The ability of nano-forms of plant-derived
nanoparticles to cross the blood–brain-barrier was
previously suggested to be mediated by nano-silica or
nano-silicon vehicles and biological amplifiers that would
be augmenting the Cocculus nanoparticles (Demangeat,
2010; Dhawan et al., 2011; Mathew et al., 2012).
Previous animal studied have demonstrated the biological
effects of various homeopathic treatments for SD (Ruiz-
Vega et al., 2002, 2005). Nunes Junior et al. (1994)
used the Multiple Platform Method (MPM) as suggested
animal model to induce SD. This method was practiced
in several studies (Suchecki et al., 1998; Suchecki and
Tufik, 2000; Yang et al., 2010) and proved to be
efficient in inducing Rapid-eye-movement (REM)
deprived rats, generating less stress vis-a
`-vis other
acceptable SD methods (Rechtschaffen and Bergmann,
2002; Machado et al., 2006). In rats, chronic SD was
shown to cause death after 16–21 days from the onset
of deprivation. In comparison, food deprivation was
shown to cause death after 17–19 days (Orzel-
Gryglewska, 2010). On the other hand, acute SD was
not observed to cause destructive effects either on cells,
or vital organs (Orzel-Gryglewska, 2010). However, it
increases energy outlay (Martins et al., 2010), with a
tendency to decrease both leptin (Rosa Neto et al.,
2010) and the anabolic testosterone (Wu et al., 2011;
Dattilo et al., 2012). Moreover, SD increases serum
serotonin (Hipolide et al., 2005) as well as its
extracellular concentrations in the hippocampus. It
elevates corticosterone level (Bodosi et al., 2004; Tiba
et al., 2008; Galvao Mde et al., 2009; Martins et al.,
2010; Rosa Neto et al., 2010; Wu et al., 2011), while
this elevation is independent of the stress response
(Galvao Mde et al., 2009; Mongrain et al., 2010) and is
suggested to occur due to changes in circadian rhythm
(Tartar et al., 2009) and metabolic homeostasis (Dattilo
et al., 2012).
When comparing an animal model to humans, one
must take into consideration physiological as well as
brain growth trajectories, especially when rat and human
maturation cannot be compared as linear timeline
development (Erecinska et al., 2004; Quinn, 2005).
A systematic human study of the effects of SD is ethically
limited. Moreover, controlling, mediating and moderating
potential artifact variables are especially difficult when
examining the long-term effects of SD on humans’
cognitive functioning. Thus, it is customary to use an
animal model in order to investigate the long-term
effects of SD. Similar to humans, in rats sleep is an
important physiological process regarded as a basic
need for functioning and survival (Everson, 1995). Many
studies have proposed different deprivation paradigms,
including partial chronic (Machado et al., 2006) or acute
(Suchecki et al., 1998;Schwierin et al., 1999;Suchecki
and Tufik, 2000) deprivations, with similar effectiveness.
While considering treatment of SD, normal
spontaneous recovery must be taken into account
(Schwierin et al., 1999;Orzel-Gryglewska, 2010). Our
current study aims are to explore the effects of acute
SD during early adulthood, focusing on short-term
behavioral effects as well as on long-term hormonal
modulations. Specifically, we aimed to examine the
short- (post 1-, 6-, 12- and 24-h) and long-term (post
14 days) effects of acute 48 h SD on fatigue, attention,
and motor learning. Moreover, we aimed to examine the
outcomes of cocc 30c treatment on both behavioral and
hormonal modulations following SD.
Forty-four male Wistar rats (weighing between 200 and
220 gr) were purchased from Harlan (Jerusalem, Israel)
and were given 7 days of acclimation in the institutional
animal housing facility. Rats were housed four per cage
cm). Room temperature was
maintained at 23 ± 1 °C with 67% humidity at 12:12-
day/night cycle (lights on at 0600). Food and water
access were allowed ad libitum. This study was
conducted in strict accordance with the
recommendations of the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health.
The protocol was approved by the Institutional Animal
Care and Use Committee. All efforts were made to
minimize animal suffering.
Rats were randomly assigned into four groups: Naive
(n= 12), Naive rats that were treated with cocc 30c
(Naive Treated; n= 12), sleep deprived (SD; n= 16),
and sleep deprived + Treatment (SD treated; n= 16).
2 S. Zubedat et al. / Neuroscience 253 (2013) 1–8
Starting on Postnatal day (PND) 70 rats were subjected to
SD for 48 h, using MPM (method as described below)
(Suchecki and Tufik, 2000). cocc 30c or placebo was
given Per Os starting immediately after baseline tests
(i.e. 1 h post SD), and subsequently every 3 h during
24 h post SD. One hour after SD, prior to treatment, rats
were tested in the startle box for baseline evaluation.
Additional re-tests were held at 6, 12 and 24 h, as well
as at 14 days post SD. Pre-pulse inhibition (PPI) was
examined at 12 and 24 h. Motor learning test in the
Rotor-rod was performed starting 48 h post-SD (for total
of 4 days of training). Rats were handled prior to any
manipulation. A description of the procedure is shown in
MPM. This method is based on the muscle atonia
characteristics of REM sleep. In short, this method
[modified from (Suchecki and Tufik, 2000)] includes
placing the animal on top of a narrow platform located
inside a water tank that allows the animal to lie down.
As muscle atonia occurs, the rat falls into the water and
is deprived of REM sleep.
In the current study, the water tank (120 cm in
diameter, 60 cm in height) contained 30 narrow
platforms (6.5 cm in diameter) placed inside. Sixteen
animals (in each round) were placed on top of the
platforms, allowing them to move around. The tank was
filled with water (6 cm) until 1 cm below the platform,
thus allowing the rat to climb back in case of falling into
the water. Exposure to the MPM began at 0800, and
animals remained inside the tank for 48 h. In order to
verify the effectiveness of MPM, rats were videotaped
by an infra-red Ikegami camera. Room temperature was
maintained at 23 ± 1 °C. Food and water were provided
ad libitum. All rats accomplished the entire study (no
deaths occurred).
cocc 30c treatment. cocc 30c is a homeopathic
remedy made of highly diluted tincture of Anamirta
cocculus seeds powder, first prepared and published by
Hahnemann in 1821 (Hahnemann, 1821). The plant
originates from the coast of Malabar, India, and Ceylon.
The tincture is prepared by macerating the powdered
seeds. The Cocculus preparation: The Cocculus was
prepared in ‘helios pharmacy (UK)’. It was sent in a
bottle containing 99% ethanol and went through 40
succussions in 90% ethanol, by manual shaking. Sixty
globules were wetted in the solution and dried for
several minutes. Following this procedure, cocc 30c
made from sucrose immersed in a solution of Cocculus
indicus 30c (40 mg dissolved in 1 ml tap water), was
administered Per Os every three hours immediately
after baseline tests for a period of 24 h. Placebo
treatment was 1% sucrose solution similar to the
hedonic effect caused by the sweet taste of cocc 30c.
The experiment was carried out in a random double-
blind design i.e. Experimenter 1 (Dr. Freed) prepared
the cocc 30c or placebo treatments in two identical
tubes (labeled ‘‘A’’ or ‘‘B’’) and delivered to the principal
investigator (Dr. Avital) the tubes on the day of
experiment. The experimenters (all the authors except
Dr. Avital and Freed) got the labeled tubes. Across total
number of rats, a randomized assignment to one of four
groups was made until accomplishing a total assignment
of 16 rats in the treated groups and 12 in the Naive and
Naive Treated rats. cocc 30c or placebo treatments
were revealed after the results analysis.
Behavioral tests
PPI and startle response. The test is held in a
ventilated soundproof box (Campden instruments, UK)
and aims to examine Startle response as well as the
function of the sensorimotor gating.
The session (a total of 90 trials) begins with three min
acclimation period with a 57-dB background white noise
level that is delivered continuously throughout the test
session. To evaluate the startle response, each of the
first ten trials consist of a single 40-ms 120-dB ‘‘pulse
alone’’ startle stimuli (Inter-Trial-Interval (ITI) 1 min). The
rest of the 80 trials (10 s ITI) consist of random delivery
of: 10 ‘‘no stimuli’’ trials, during which no stimuli are
delivered, fourteen ‘‘pre’’ stimuli (at 59, 61, 65, 69, 73,
78 or 85 dB), and 56 ‘‘pre-pulse’’ trials that include a
single 120 dB pulse preceded (80 ms interval) by a
20 ms pre pulse of 2, 4, 8, 12, 16, 21 or 28 dB above
background (i.e., 59, 61, 65, 69, 73, 78 or 85 dB).
Finally, PPI was calculated as the percent of the
habituated response as follows: [100 (max response
to ‘‘pre-pulse’’ trial/max response to ‘‘pulse alone’’
trial 100)] (Avital et al., 2011; Ram et al., 2013).
Rotor-rod. The rotor-rod (San Diego instruments, San
Diego, CA, USA) apparatus is used to assess motor
functions, motor learning, coordination, and equilibrium.
It is made of black lusterless Perspex, comprised of a
rotating rod with four lanes separated by opaque black
Perspex. The apparatus contains a sawdust cabin
(45.7 cm height) for safe landing and automatic
recording of latencies (in sec.) via red-beams system.
Each learning session consists of four daily trials, on
four consecutive days. Rats are acclimated to the
apparatus for 30 s on the first day. Each trial starts at
five rpm for 15 s with constant acceleration at 0.1 rpm
per second (max speed 50 rpm after 460 s).
Corticosterone, testosterone, serotonin and leptin
In order to avoid circadian variability, all samples were
collected between 1100 and 1200, when plasma
hormones’ concentration is relatively low. Blood
samples were taken immediately after decapitation and
centrifuged (2000gat 4 °C for 20 min), serum was
collected and stored at 80 °C until assayed. Serum
corticosterone and testosterone levels were assessed
using commercial enzyme-linked immunosorbent assay
(ELISA) kits (AssayPro, St. Charles, MO, USA)
according to the manufacturer’s instructions.
Serum leptin was quantified using specific enzyme
immunoassay (Genese
, Brazil). Serotonin
concentration was determined using commercial
S. Zubedat et al. / Neuroscience 253 (2013) 1–8 3
enzyme assay kit (Diagnostic products Corp., Los
Angeles, CA, USA) (Wu et al., 2011).
Statistical analysis
A Bonferroni multiple correction analysis was performed
in order to avoid possible confounds due to the
substantial amount of testing. Data were analyzed for
statistical significance using an analysis of variance
(ANOVA) for mixed design, with group as between-
subject factor and test timing or pre-pulse intensity as
within-subject factor. In order to further explore the main
effects, we used a one-way ANOVA followed by a Post-
Hoc Tukey test. A result was considered significant
when P< 0.05. All tests were calculated as two-tailed
using SPSS V17.0. Results are presented as
means ± standard error of the means (SEM).
A Bonferroni multiple correction analysis was performed
in order to avoid possible confounds and the substantial
amount of testing after the SD that may bound to affect
the recovery sleep. The analysis yielded no effect, thus,
it excludes the possible confound that may affect the
sleep recovery.
Startle response
In order to examine short- and long-term progression in
startle response (Fig. 2), an ANOVA for mixed design
was carried-out with group as between-subject factor
and test time as within-subject factor. The results
indicate a significant effect for test time
[F(4,17) = 39.96, P< 0.0001] as well as between
groups [F(3,20) = 7.2, P< 0.002]. Post-hoc Tukey
tests indicate a significant decrease in startle response
for both SD and SD-treated groups compared with naive
rats, at baseline (P< 0.0001), post-6 h (P< 0.0001)
and post-12 h (P< 0.01). Twenty-four hours post SD all
groups showed similar and low startle response that
probably reflects habituation and/or floor effect. Finally,
14 days post SD, while naive- and SD-treated rats
showed a recovery (from the alleged habituation) to the
naive rats’ startle response, the SD rats showed a long-
term increased startle response compared with naive
(P< 0.007), naive-treated (P< 0.0001) and SD-treated
rats (P< 0.0001).
PPI 12 h after SD (Fig. 3): a significant difference was
found along various pre-intensities [F(6, 47) = 102.97,
P< 0.0001] as well as between the groups
[F(3,52) = 4.53, P< 0.007]. Overall, a clear tendency
of impaired PPI was observed following SD.
Interestingly, the treatment with cocc 30c led to a
beneficial effect on PPI performance. Specifically, a
significant effect was found at pre-intensity 61 dB
between SD-treated and SD (P< 0.0001), naive
(P< 0.044) and naive-treated (P< 0.001) rats.
Regarding pre-intensities 65 dB and 69 dB cocc 30c led
to improved PPI compared with SD (P< 0.001;
P< 0.025, respectively).
PPI 24 h post SD (Fig. 3): similarly to 12 h post-SD
test, a significant effect was observed along different
pre-intensities [F(6,47) = 115.97, P< 0.0001] as well
as between the groups [F(3, 52) = 11.47, P< 0.0001].
The treatment with cocc 30c clearly led to elevated PPI
performance. In particular, SD-treated rats have
exhibited a significant increase at pre-intensity 61 dB,
compared with both SD (P< 0.0001) and naive
(P< 0.01) rats. Following SD, the treatment cocc 30c
led to improved PPI at pre-intensities 65 dB
(P< 0.0001), 69 dB (P< 0.0001), 73 dB (P< 0.001),
78 dB (P< 0.001) and 85 dB (P< 0.005), compared
with SD group.
A significant motor learning effect (Fig. 4) was
found during 4 days of training [F(3, 132) = 49.72,
Fig. 1. A schematic description of the experimental procedure. A description of experimental time line and postnatal days, along which the exposure
to sleep deprivation, treatment, behavioral and hormonal examinations took place.
4 S. Zubedat et al. / Neuroscience 253 (2013) 1–8
P< 0.0001] with significant difference between the
groups [F(3,134) = 39.74, P< 0.0001]. Moreover, a
significant effect was found for the group day
interaction [F(9,392) = 2.43, P< 0.011]. Post-hoc
Tukey test indicated a significantly enhanced
performance in the SD-treated group, compared with
SD (P< 0.0001), naive (P< 0.0001) and naive-treated
(P< 0.028), on the first day of motor learning. This
enhanced performance for the treated SD was also
significant (P< 0.0001) on the second day compared
with SD, naive and naive-treated. On the third day SD-
treated performed better compared with naive
(P< 0.004) and SD (P< 0.0001) groups. This
tendency remains on the fourth day of motor learning,
as the SD-treated rats performed better than SD rats
(P< 0.0001).
A significant group effect on corticosterone serum
(Fig. 5A) levels [F(3,54) = 50.72, P< 0.0001] was
observed. Post-hoc Tukey test indicated a significant
elevation in corticosterone following SD (P< 0.0001)
compared with naive and naive-treated groups.
However, the treatment with cocc 30c significantly
moderated this elevation toward (P< 0.0001; compared
with SD group).
A significant group effect on testosterone serum (Fig. 5B)
level [F(3,51) = 13.35, P< 0.0001] was observed. Post-
hoc Tukey test indicated a significant decrease in
testosterone level following SD (P< 0.0001), while cocc
30c treatment significantly restored this decrease
(P< 0.013; compared with SD group).
A significant group effect on serotonin serum (Fig. 5C)
level [F(3,51) = 23.46, P< 0.0001] was observed.
Post-hoc Tukey test indicated a significant elevation
following SD (P< 0.0001), while cocc 30c treatment
moderated the observed elevation (P< 0.0001;
compared with SD group).
A significant group effect on leptin serum (Fig. 5D) level
[F(3,51) = 118.15, P< 0.0001] was observed. Post-
hoc Tukey test indicated a significant decrease in leptin
level following SD (P< 0.0001), while cocc 30c
treatment significantly repaired the latter decrease to a
moderated level (P< 0.0001; compared with SD group).
Attentional and motor reactivity
Both treated and untreated SD groups showed hypo-
responsiveness to the startle stimulus. Though naive
rats showed habituation along the four time points that
were examined (i.e. baseline, 6, 12 and 24 h post-SD),
the lack of this habituation in the SD groups may be
attributed to floor effect. Fourteen days post SD, all
groups showed a recovery from the alleged habituation,
similar to the naive group. However, the SD group
Fig. 2. Startle reflex. A significant decrease in startle response was observed in both SD and SD-treated groups compared with naive rats, at
baseline, post-6 h and post-12 h. Fourteen days post SD, while naive- and SD-treated rats showed a recovery (from the alleged habituation) to the
naive rats startle response, the SD rats showed a long-term increased startle response compared with naive with SD-treated rats (
P< 0.0001;
P< 0.01).
Fig. 3. Pre-pulse inhibition (PPI). The treatment with cocc 30c led to
a beneficial effect on PPI performance measured both 12- and 24-h
post SD (
P< 0.0001;
P< 0.001;
P< 0.005;
P< 0.025).
S. Zubedat et al. / Neuroscience 253 (2013) 1–8 5
showed a significant increased startle response.
Considering the noise during the MPM procedure in
which 16 rats stayed together in the same arena, one
may postulate that the effects of ambient noise in the
different environments can explain the hypo-
responsiveness to the startle stimulus (Baldwin et al.,
2006). This elevation in startle response observed in all
groups, may reflect the extensive test procedure that
the rats underwent, together with evidence that startle
response is suggested to be age-dependent (Weiss
et al., 2001).
Previously it has been shown that SD in humans
impairs attention ability (Alhola and Polo-Kantola, 2007;
McCoy and Strecker, 2011). Specifically, acute SD
(such as total 24–48-h deprivation) was previously
shown to impair the performance in attention tasks
(Blagrove et al., 1995; Kendall et al., 2006; Bocca and
Denise, 2006). Thus, we aimed to test the sensorimotor
gating utilizing the PPI test, which relates to attention
processes (Avital et al., 2011; Ram et al., 2013). SD led
to impaired PPI ability 12 h post deprivation.
Surprisingly, the treated rats showed an immediate
effect, with superior PPI ability compared with all other
groups. Twenty-four hours post SD, though there was
no significant difference in PPI performance between
the control and the SD groups (presumably due to
spontaneous recovery from SD), yet the SD-treated rats
showed a better PPI performance compared with all
other groups. The striatum is considered to be the
relevant region for sensorimotor gating (Moore et al.,
2006). Indeed, Lim et al. (2011) have recently found a
significant decrease in dopamine receptor1 (D1R), no
change in D2R, and a significant increase in D3R
binding in the striatum, following SD. This pattern was
not observed following stress, thus suggesting to be a
specific remodeling of dopaminergic circuits after SD.
Considering dopamine as the core neurotransmitter
involved in attention processes in both the prefrontal
cortex and striatum (Moore et al., 2006; Kumari et al.,
2008; Molina et al., 2009), we postulate that the
beneficial effect of cocc 30c on PPI performance,
immediately following SD, may be associated to the
increased binding to D3R in the striatum.
Fig. 5. Hormonal serum levels. Fourteen days after the exposure to sleep deprivation, a significant increase in both corticosterone (A) and serotonin
(B) serum level was observed in the SD group. However, the treatment with cocc 30c had a long-term beneficial effect, manifested in a significant
decrease of both corticosterone and serotonin. Moreover, sleep deprivation led to decreased serum levels of Testosterone (C) and Leptin (D).
Interestingly, cocc 30c treatment recovered these decrements (
P< 0.0001;
P< 0.013).
Fig. 4. Motor learning in the rotor-rod test. The treatment with cocc
30c led to a better motor learning ability (starting 48 h post SD) across
all 4-days learning SD (
P< 0.0001).
6 S. Zubedat et al. / Neuroscience 253 (2013) 1–8
Taken together, the behavioral tests have indicated
significant short-term effects of SD. The treatment with
cocc 30c remedy seems to improve these short-term
deteriorating effects
Hormonal modulations
In order to explore whether SD has a long-term ‘‘covert’’
hormonal effects while the ‘‘overt’’ behavior effects are
recovered, we examined corticosterone, testosterone,
serotonin and leptin serum levels, 14 days post SD. The
notion on ‘‘covert’’ hormonal and ‘‘overt’’ behavioral
effects of SD is supported by Lopez-Rodriguez et al.
(2003) report that extracellular concentration of
serotonin remained high at the end of SD recovery day
period, though displaying normal amount of sleep.
Following SD, corticosterone and serotonin serum
levels were elevated and the treatment with cocc 30c
recovered these elevations. Moreover, testosterone and
leptin decreased following SD, and cocc 30c treatment
moderated this decline. Our findings are in line with a
previous study (Wu et al., 2011), which reported a
reduction of serum testosterone, and elevated levels of
serotonin and corticosterone, following SD. Considering
the inverse secretion relation between testosterone and
5-hydroxytryptamine (5-HT) (Frungieri et al., 2002), it is
plausible that the reduction in testosterone level is due
to 5-HT inhibition of testosterone production, or vice
versa. Similarly to our 48-h SD effect on corticosterone
and leptin levels, it was previously found that 96 h of SD
led to increased corticosterone as well as decreased
leptin serum levels (Rosa Neto et al., 2010). Moreover,
Koban and Swinson (2005) showed that leptin
decreased after SD and remained low following twenty
days of recovery.
Taken together, the treatment with cocc 30c seems to
restore the deteriorating effects of 48 h of SD on
attention and motor learning abilities. Examining the
long-term effects of SD, cocc 30c dramatically
recovered the hormonal alterations observed.
Bioavailability and biological activity of nano-forms of
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Chuaughule (2013) have shown that succussions can
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(Accepted 13 August 2013)
(Available online 22 August 2013)
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... In healthy subjects, experimentally controlled 24 h sleep deprivation reduced the PPI response in a similar manner to psychotic patients, and therefore, PPI response was suggested as a marker to understand psychotic event pathogeny after sleep deprivation [44]. Sleep deprivation, i.e. 72 h REM sleep deprivation, was shown to reduce the PPI response also in rats, suggesting the crucial role of proper REM sleep in the modulation of PPI response [45][46][47][48]. The impairment was reversed by the use of antipsychotics such as haloperidol, clozapine and risperidon, and REM sleep deprivation was recommended as a partial symptomatic animal model of schizophrenia, due to the similar symptomatic spectrum including the PPI impairment endophenotype [45]. ...
... In this study, 72 h REM sleep deprivation was preferred as it was previously shown to reduce the PPI response in rats [45][46][47][48], and REM sleep deprivation was recommended as a partial symptomatic animal model of schizophrenia, due to the similar symptomatic spectrum including the PPI impairment endophenotype [45]. Previous studies also mentioned a possible link between orexinergic system and neurodevelopmental disorders, such as schizophrenia [25][26][27][28]. ...
Orexin is a novel neurotransmitter released from lateral hypothalamus, that is a crucial modulator in sleep/wakefulness system. Recent studies also suggest its possible role in the neurodevelopmental disorders, such as schizophrenia. Our study consists of two experiments, where we investigate the effect of orexin A (OXA), one of two isoforms of orexin that can pass blood brain barrier, on the prepulse inhibition of acoustic startle reflex. The first experiment tested the effect of OXA on PPI response of non-sleep-deprived rats via intraperitoneal injection 30 min before testing. Our results show that 40 μg/kg OXA attenuates PPI% at 78 dB and 86 dB prepulse intensities. The second experiment utilized 72-hour REM sleep deprivation, as a model for sleep-deprivation-induced impairment of PPI response. Here, we tested the effect of OXA on PPI% of sleep-deprived rats via intraperitoneal injection at the last 30 min of sleep deprivation, testing for PPI immediately afterwards. Our results showed that (1) sleep deprivation attenuates the PPI% at 74 dB, 78 dB and 86 dB prepulse intensities and (2) 10 μg/kg OXA completely restores the impaired PPI% at 78 dB only, where the highest PPI% impairment was observed. These results suggest that orexin A modulates PPI response in rats in a dose-dependent manner, oppositely for non-sleep-deprived and sleep-deprived rats, and a more detailed investigation for the etiology of this effect should follow.
... The amount of 5,7-DHT administered in these experiments caused extensive loss of 5-HT neurons in the forebrain, as shown by a reduction of 92.4% ± 1.05% (mean ± SEM, n = 54) in striatal content of 5-hydroxyindole acetic acid, the major metabolite of 5-HT. 43 In a separate experiment, a clear difference was seen between sensor responses to breath collected from a training set of Tg rats and their WT controls with a sensitivity of 100%, specificity of 90%, and accuracy of 94% ( Figure 4). A different set of sensors was required for best separation between A53T rats and controls from that which gave best separation in samples from dopaminergic-and serotonergic-lesioned rats (see Methods), and the control rats were from different suppliers, so data in Figures 3 and 4 cannot be compared. ...
... Rats were acclimated to the apparatus for 30 s on the first day. Each trial starts with 5 rpm speed for 15 s followed by an acceleration of 0.1 rpm per second (max speed 50 rpm after 460s), and the latency until the rat falls from the rod was measured as previously described 43 Blood and Brain Tissue VOCs Analysis. Rats were anesthetized using 100 mg/kg Inactin (thiobutabarbital sodium 100 mg/kg, Sigma); then the posterior vena cava was exposed, saline with heparin (0.4 mL with 0.05 mL of heparin 5000 units) was injected into the abdominal vena cava using a 1 mL sterile disposable plastic syringe, and 1 mL of venous blood was withdrawn into the syringe. ...
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Early diagnosis of Parkinson’s disease (PD) is of great importance due its progressive phenotype. Neuroprotective drugs could potentially slow down disease progression if used at early stages. Previously, we have reported an altered content of volatile organic compounds (VOCs) in the breath of rats following a 50% reduction in striatal dopamine (DA) content induced by 6-hydroxydopamine. We now report on the difference in the breath-print and content of VOCs between rats with mild and severe lesions of DA neurons, serotonergic neuronal lesions, and transgenic (Tg) rats carrying the PD-producing A53T mutation of the SNCA (alpha-synuclein) gene. The Tg rats had an increased content of 3-octen-1-ol and 4-chloro-3-methyl phenol in blood, while in brain tissue, hexanal, hexanol and 2,3-octanedione were present in controls but absent in Tg rats. Levels of 1-heptyl-2-methyl cyclopropane were increased in brain tissue of Tg rats. The data confirm the potential of breath analysis for detection of human- idiosyncratic as well as autosomal dominant PD.
... A. cocculus NPs in cocc 30c, in a homeopathic formulation Preclinical Improved attention and motor functions in sleep-deprived rats [44] Solanum tuberosum L. ...
Full-text available
Citation: Rajagopal, M.; Paul, A.K.; Lee, M.-T.; Joykin, A.R.; Por, C.-S.; Mahboob, T.; Salibay, C.C.; Torres, M.S.; Guiang, M.M.M.; Rahmatullah, M.; et al. Phytochemicals and Nano-Phytopharmaceuticals Use in Skin, Urogenital and Locomotor Disorders: Are We There? Plants 2022, 11, 1265. plants11091265 Academic Editors: Konstantinos Gardikis and Ioannis Mourtzinos
... The Rotarod test is widely used for measuring motor coordination and motor skill learning in rodents (Eltokhi et al., 2021;Shiotsuki, 2010;Deacon, 2013). Wistar rats show motor learning ability with increasing latency to fall during a 4-d rotarod test (Zubedat, 2013), however in our study, SD rats from the control group did not show a significant improvement in the latency to fall throughout the 4-day learning period. Different strains of animals (SD vs. Wistar rats) might explain the difference in these findings since many factors including strain, sex and body weight affect rodents' performance in the Rotarod test (Eltokhi et al., 2021;Feyissa, 2017). ...
Static magnetic fields (SMF) have neuroprotective and behavioral effects in rats, however, little is known about the effects of SMF on cognition, motor function and the underlying neurochemical mechanisms. In this study, we focused on the effects of short-term (5~10d) and long-term (13~38d) SMF exposure on selective attention and motor coordination of rats, as well as associated alterations in expression level of neuroplasticity-related structural proteins and cryptochrome (CRY1) protein in the cortex, striatum and ventral midbrain. The results showed that 6 d SMF exposure significantly enhanced selective attention without affecting locomotor activity in open field. All SMF exposures non-significantly enhanced motor coordination (Rotarod test). Neurochemical analysis demonstrated that 5d SMF exposure increased the expression of cortical and striatal CRY1 and synapsin-1 (SYN1), striatal total synapsins (SYN), and synaptophysin (SYP), growth associated protein-43 (GAP43) and post-synaptic density protein-95 (PSD95) in the ventral midbrain. Exposure to SMF for 14d increased PSD95 level in the ventral midbrain while longer SMF exposure elevated the levels of PSD95 in the cortex, SYN and SYN1 in all the examined brain areas. The increased expression of cortical and striatal CRY1and SYN1 correlated with the short-lasting effect of SMF on improving selective attention. Collectively, SMF’s effect on selective attention attenuated following longer exposure to SMF whereas its effects on neuroplasticity-related structural biomarkers were time- and brain area-dependent, with some protein levels increasing with longer time exposure. These findings suggest a potential use of SMF for treatment of neurological diseases in which selective attention or neuroplasticity is impaired.
... A. cocculus NPs in cocc 30c, in a homeopathic formulation Preclinical Improved attention and motor functions in sleep-deprived rats [44] Solanum tuberosum L. ...
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Nanomedicines emerged from nanotechnology and have been introduced to bring advancements in treating multiple diseases. Nano-phytomedicines are synthesized from active phytoconstituents or plant extracts. Advancements in nanotechnology also help in the diagnosis, monitoring, control, and prevention of various diseases. The field of nanomedicine and the improvements of nanoparticles has been of keen interest in multiple industries, including pharmaceutics, diagnostics, electronics, communications, and cosmetics. In herbal medicines, these nanoparticles have several attractive properties that have brought them to the forefront in searching for novel drug delivery systems by enhancing efficacy, bioavailability, and target specificity. The current review investigated various therapeutic applications of different nano-phytopharmaceuticals in locomotor, dermal, reproductive, and urinary tract disorders to enhance bioavailability and efficacy of phytochemicals and herbal extracts in preclinical and in vitro studies. There is a lack of clinical and extensive preclinical studies. The research in this field is expanding but strong evidence on the efficacy of these nano-phytopharmaceuticals for human use is still limited. The long-term efficacy and safety of nano-phytopharmaceuticals must be ensured with priority before these materials emerge as common human therapeutics. Overall, this review provides up-to-date information on related contemporary research on nano-phytopharmaceuticals and nano-extracts in the fields of dermatological, urogenital, and locomotor disorders.
... Interactúa con varias disciplinas, abarcando la biología celular, la filosofía, la física nuclear, la inmunología, la neurociencia, la patología, la psicología, la toxicología, la sociología, la termoquímica, etcétera [156][157][158][159][160][161] . Ahora bien, los defensores del EK dicen que los pseudocientíficos se las arreglan para interpretar cada fracaso como una confirmación 165 , que evitan responder las críticas porque las perciben siempre como un ataque 166 , y que nunca publican resultados contrarios 167 . ...
Homeopathy is a controversial field of study, so much that it has even been considered by some philosophers of science as a fraud. The question of homeopathy as pseudoscience can be approached with at least two strategies: a) evaluating the main arguments of so-called skeptics of “pseudoscience”, and b) discussing the main demarcation criteria between science or pseudoscience proposed by the philosopher Mario Bunge. We take both approaches and will discuss some points to favor or against homeopathy. Since the beginning, Bunge’s demarcation was considered as the most reliable “evidence” against homeopathy. A primary goal of this article is to provide some examples to illustrate the debate. At the end, this article demonstrates that the Bunge’s demarcation is an act of faith that is ultimately completely invalid in comparison to the contemporary growing body of scientific evidence. On the basis of the discussion presented in the article, the Bunge’s demarcation must not be used to provide any definitive categorizations.
... Indeed it has been well established that TSD, a stress condition, is responsible for sustained attention deficits and hormonal impairments (Maggio et al., 2013;Arnal et al., 2015Arnal et al., , 2016 with a decrease of morning levels of circulating testosterone and cortisol (Arnal et al., 2016). Circadian misalignment/Sleep restriction in humans or REM/TSD in rodents were associated with an increase of adrenal activity and/or a decrease of testosterone levels (Axelsson et al., 2005;Reynolds et al., 2012;Schmid et al., 2012;Zubedat et al., 2013) that could interfere with cognitive functions, including memory (Maggio et al., 2013). Besides, salivary α-amylase (sAA) level is considered as a useful tool for evaluating SAM activity, and was found lowered under chronic psychological stress in association with impairments in cognitive performance (Teixeira et al., 2015). ...
... Indeed it has been well established that TSD, a stress condition, is responsible for sustained attention deficits and hormonal impairments (Maggio et al., 2013;Arnal et al., 2015Arnal et al., , 2016 with a decrease of morning levels of circulating testosterone and cortisol (Arnal et al., 2016). Circadian misalignment/Sleep restriction in humans or REM/TSD in rodents were associated with an increase of adrenal activity and/or a decrease of testosterone levels (Axelsson et al., 2005;Reynolds et al., 2012;Schmid et al., 2012;Zubedat et al., 2013) that could interfere with cognitive functions, including memory (Maggio et al., 2013). Besides, salivary α-amylase (sAA) level is considered as a useful tool for evaluating SAM activity, and was found lowered under chronic psychological stress in association with impairments in cognitive performance (Teixeira et al., 2015). ...
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Chronic sleep restriction (CSR) induces neurobehavioral deficits in young and healthy people with a morning failure of sustained attention process. Testing both the kinetic of failure and recovery of different cognitive processes (i.e. attention, executive) under CSR and their potential links with subject’s capacities (stay awake, baseline performance, age) and with some biological markers of stress and anabolism would be useful in order to understand the role of sleep debt on human behavior. Twelve healthy subjects spent 14 days in laboratory with 2 baseline days (B1 and B2, 8h TIB) followed by 7 days of sleep restriction (SR1-SR7, 4h TIB), 3 sleep recovery days (R1-R3, 8h TIB) and 2 more ones 8 days later (R12-R13). Subjective sleepiness (KSS), maintenance of wakefulness latencies (MWT) were evaluated 4 times a day (10:00, 12:00 a.m. and 2:00, 4:00 p.m.) and cognitive tests were realized at morning (8:30 a.m.) and evening (6:30 p.m.) sessions during B2, SR1, SR4, SR7, R2, R3 and R13. Saliva (B2, SR7, R2, R13) and blood (B1, SR6, R1, R12) samples were collected in the morning. Cognitive processes were differently impaired and recovered with a more rapid kinetic for sustained attention process. Besides, a significant time of day effect was only evidenced for sustained attention failures that seemed to be related to subject’s age and their morning capacity to stay awake. Executive processes were equally disturbed/recovered during the day and this failure/recovery process seemed to be mainly related to baseline subject’s performance and to their capacity to stay awake. Morning concentrations of testosterone, cortisol and α-amylase were significantly decreased at SR6-SR7, but were either and respectively early (R1), tardily (after R2) and no recovered (R13). All these results suggest a differential deleterious and restorative effect of CSR on cognition through biological changes of the stress pathway and subject’s capacity (ClinicalTrials-NCT01989741).
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Homeopathic remedies (HRs) contain odorant molecules such as flavonoids or terpenes and can lose their efficiency in presence of some competitive odors. Such similarities, along with extreme sensitivity of the olfactory system, widespread presence of olfactory receptors over all organic tissues (where they have metabolic roles besides perception of odors), and potential direct access to the brain through olfactory nerves (ONs) and trigeminal nerves, may suggest the olfactory system as target for HRs. Recent works highlighted that HRs exist in a dual form, that is, a still molecular form at low dilution and a nanoparticulate form at high dilution, and that remnants of source remedy persist in extremely high dilutions. From the literature, both odorants and nanoparticles (NPs) can enter the body through inhalation, digestive absorption, or through the skin, especially, NPs or viruses can directly reach the brain through axons of nerves. Assuming that HRs are recognized by olfactory receptors, their information could be transmitted to numerous tissues through receptor-ligand interaction, or to the brain by either activating the axon potential of ONs and trigeminal nerves or, in their nanoparticulate form, by translocating through axons of these nerves. Moreover, the nanoparticulate form may activate the immune system at multiple levels, induce systemic various biological responses through the pituitary axis and inflammation factors, or modulate gene expression at the cellular level. As immunity, inflammation, pituitary axis, and olfactory system are closely linked together, their permanent interaction triggered by olfactory receptors may thus ensure homeostasis.
Homeopathy is one of the most widely practiced alternative systems of medicine in the world. Current scientific understanding is unable to explain its mode of action, and the therapy is often dismissed by detractors who claim—despite growing evidence to the contrary— that it is ineffective. While homeopathy's philosophical foundations and the nature of its medicines differ markedly from those of its mainstream counterpart, biomedical researchers are nevertheless employing conventional methods to study it—including lab-based animal experimentation. This article considers the implications of using violent methodology to investigate a gentle system of medicine founded on treating the individual and on doing no harm.
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To determine whether noise-induced stress disrupts the intestinal mucosa of laboratory rats, one group of 8 rats ('noise' rats) was subjected to 15 min of white noise (90 dB) daily for 3 wk. Another group ('quiet' rats) was housed for 3 wk in an acoustically similar room but with no additional noise. A 3rd group ('recovery' rats) was housed in the noise room for 3 wk and then in the quiet room for a further 3 wk. The ilea were fixed for microscopy. Villi adjacent to Peyer patches showed significantly more degranulated mast cells (mean standard error of the mean, 3.95 +/- 0.80 versus 0.35 +/- 0.29, respectively) and eosinophils (mean standard error of the mean, 9.46 +/- 0.44 versus 4.58 +/- 0.38) per villus section in noise rats than in quiet rats. Similar results were obtained with rooms reversed, to account for any differences in room characteristics. The mean width of villus laminar propria was significantly greater in noise rats than quiet rats, suggesting edema. In addition, mucosal epithelial cells of noise rats were often separated, sometimes detaching from the basement membrane, whereas those of quiet rats were intact. Behaviorally, noise rats exhibited significantly more grooming and rearing than quiet rats. Compared with noise rats, recovery rats showed no reduction in mast cell degranulation or mean width of villus lamina propria, but there were increased numbers of secreting goblet cells in villi adjacent to Player patches and some recovery of epithelial integrity.
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This paper presents evidence-based results of clinical trial on the use of nanosized particles in homoeopathic Terminalia arjuna tincture. Dynamic light scattering technique demonstrates that homoeopathic tincture is a mixture of particles varying from micron to nanometre sizes. The process of succussion, homogenization and filtration brings down the particles of source material to nanosize as a ‘top-down’ process. As compared to the conventionally used micron-sized T. arjuna powder, the results show significant clinical improvement due to the presence of nanoparticles in tincture by the above method. Similar results were reconfirmed on experiments with bacterial cultures. From clinical trials, it has been confirmed that the dose of medicine required by the patient is significantly reduced with the reduction in particle size. No toxicity was observed even after giving medicine for more than 8 weeks. Size-dependent sensitivity of the drug can help to tackle the problem of drug resistance in bacteria.
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Increased vulnerability to psychiatric disorders, such as schizophrenia, has been associated with higher levels of stress. In the early development of the central nervous system, changes in function of glutamatergic N-Methyl-D-aspartate (NMDA) receptors can possibly result in the development of psychosis, cognitive impairment and emotional dysfunction in adulthood. Thus, in this study we examined the behavioural consequences of the exposure of male rats to chronic stress (postnatal days 30-60) and ketamine administration (postnatal days 41-45); both during a sensitive developmental time window. We found that the locomotor activity of both ketamine and ketamine+chronic stress groups was significantly higher compared with that of the control rats. In contrast, the locomotor activity of the chronic stress group was significantly lower compared with all other groups. Examining anhedonia in the sucrose preference test we found a significantly decreased sucrose intake in both ketamine+chronic stress and the chronic stress groups compared with the control rats. No significant differences were observed in sucrose intake between the control and the ketamine group. The object recognition test revealed that the attention to the novel object was significantly impaired in the ketamine+chronic stress group. Similarly, the ketamine+chronic stress group showed the poorest learning ability in the eight-arm radial maze, starting on the 8th day. Finally, throughout the different pre-pulse intensities, the ketamine+chronic stress group showed impaired PPI compared with all other groups. The results indicate that the combination of prepubertal onset of chronic stress and ketamine may serve as a valid novel animal model for schizophrenia-like symptoms.
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Background This paper proposes a novel model for homeopathic remedy action on living systems. Research indicates that homeopathic remedies (a) contain measurable source and silica nanoparticles heterogeneously dispersed in colloidal solution; (b) act by modulating biological function of the allostatic stress response network (c) evoke biphasic actions on living systems via organism-dependent adaptive and endogenously amplified effects; (d) improve systemic resilience. Discussion The proposed active components of homeopathic remedies are nanoparticles of source substance in water-based colloidal solution, not bulk-form drugs. Nanoparticles have unique biological and physico-chemical properties, including increased catalytic reactivity, protein and DNA adsorption, bioavailability, dose-sparing, electromagnetic, and quantum effects different from bulk-form materials. Trituration and/or liquid succussions during classical remedy preparation create “top-down” nanostructures. Plants can biosynthesize remedy-templated silica nanostructures. Nanoparticles stimulate hormesis, a beneficial low-dose adaptive response. Homeopathic remedies prescribed in low doses spaced intermittently over time act as biological signals that stimulate the organism’s allostatic biological stress response network, evoking nonlinear modulatory, self-organizing change. Potential mechanisms include time-dependent sensitization (TDS), a type of adaptive plasticity/metaplasticity involving progressive amplification of host responses, which reverse direction and oscillate at physiological limits. To mobilize hormesis and TDS, the remedy must be appraised as a salient, but low level, novel threat, stressor, or homeostatic disruption for the whole organism. Silica nanoparticles adsorb remedy source and amplify effects. Properly-timed remedy dosing elicits disease-primed compensatory reversal in direction of maladaptive dynamics of the allostatic network, thus promoting resilience and recovery from disease. Summary Homeopathic remedies are proposed as source nanoparticles that mobilize hormesis and time-dependent sensitization via non-pharmacological effects on specific biological adaptive and amplification mechanisms. The nanoparticle nature of remedies would distinguish them from conventional bulk drugs in structure, morphology, and functional properties. Outcomes would depend upon the ability of the organism to respond to the remedy as a novel stressor or heterotypic biological threat, initiating reversals of cumulative, cross-adapted biological maladaptations underlying disease in the allostatic stress response network. Systemic resilience would improve. This model provides a foundation for theory-driven research on the role of nanomaterials in living systems, mechanisms of homeopathic remedy actions and translational uses in nanomedicine.
We investigated 20MHz water proton NMR longitudinal (T1) and transverse (T2) relaxation in ultrahigh dilutions (range 10−7M–10−47M) of a mixture of silica–lactose (Sil/Lac) in various media (water, 0.15M NaCl, 0.15M LiCl) and in various containers (glass, polyethylene). The samples were prepared by iterative centesimal dilutions under vigorous agitation and rigorously controlled laboratory conditions. Water and salt media were similarly and simultaneously treated, as controls. No significant effect on relaxation times was induced by the iterative dilution/agitation process in pure water and salt controls. By contrast, a slight increase in T1 and decrease in T2 was observed with increasing dilution in silica–lactose solutions, resulting in a marked progressive increase in T1/T2, especially in LiCl medium, distinguishable up to the ultrahigh dilution level. Cross-correlation analyses between T1, T2 and T1/T2 managed to demonstrate opposite behaviours of controls and Sil/Lac dilutions, even in the ultramolecular range of dilution. The effect seemed dependent on the medium (LiCl>NaCl>Water), and was observed in the glassware and polyethylene series as well. After a heating/cooling cycle directly in the sealed NMR tubes, the relaxation variations observed as a function of dilution totally vanished, and the T1/T2 ratio dropped, indicating a less ordered structure. These findings were interpreted in terms of nanosized superstructures with motional correlation time greater than 5.10−9s, nucleated around the solute, and composed of water, ions and nanobubbles generated during the vigorous mechanical process. Incidentally, a striking catalytic enhancement of silica leaching was observed during the preparation of silica–lactose dilutions in glassware; but this did not influence the NMR relaxation results.
Extreme dilutions especially, homeopathic remedies of 30c, 200c and higher potencies are prepared by a process of serial dilution of 1:100 per step. As a result, dilution factors of 10^60, 10^400 or even greater, are achieved. Therefore, both, the presence of any active ingredient as well as the therapeutic efficacy of these medicines, have been contentious because existence of even traces of the starting raw materials in them is inconceivable. However, physicochemical studies of these solutions have unequivocally established the presence of the starting raw materials in nanoparticulate form even in these extreme (super-Avogadro, >10^23) dilutions. In this paper, we propose and validate a hypothesis to explain how nanoparticles are retained even at such enormous dilution levels. We show that once the bulk concentration is below a threshold level of a few nanograms/millilitre (ng/ml), at the end of each dilution step, all the nanoparticles levitate to the surface and are accommodated as a monolayer at the top. This dominant population at the air-liquid interface is preserved and carried to the subsequent step thereby forming an asymptotic concentration. Thus, all dilutions are only apparent, and not real in terms of the concentrations of the starting raw materials.
In four sleep loss experiments we aimed, first, to compare performance during long-term sleep reduction with performance during short-term total sleep deprivation, and second, to measure the effects of both methods of sleep loss on ability to ignore distracting irrelevant stimuli, using a finding embedded figures test (FEFT). Logical reasoning, auditory vigilance and finding embedded figures tasks were shown to be significantly sensitive to one night's sleep deprivation. However, in one sleep reduction study subjects reduced to a mean of 5.2 hours sleep per night for 4 weeks showed no performance deficits on logical reasoning. In a second sleep reduction study subjects reduced to a mean of 4.3 hours sleep per night for 4 nights, and subjects reduced to a mean of 5.3 hours sleep per night for 18 nights, showed no performance deficits on logical reasoning or auditory vigilance, despite their reports of severe increases in subjective sleepiness and reduced concentration. Both these sleep reduction groups, though, did show decrements on the FEFT, which we interpret in terms of dearousal increasing distractibility, which the sleep-reduced subjects could not overcome with effort, as they did with the other tests.
The methods used to induce paradoxical sleep (PS) deprivation are believed to be stressful. In the present study, two methods were compared in regard to their ability to activate the hypothalamic-pituitary-adrenal (HPA) axis. Animals were placed on multiple large (MLP) or small (MSP) platforms or on single large (SLP) or small (SSP) platforms and blood sampled at the end of a 4-day period of PS deprivation (experiment 1) or on Days 1 (short-term) and 4 (long-term) of PS deprivation (experiment 2). ACTH and corticosterone (CORT) levels were determined by RIA. The results of experiment 1 showed that all experimental animals presented increased ACTH response, compared to controls. CORT levels, however, were only elevated in MSP animals, suggesting increased adrenal sensitivity. Experiment 2 showed that ACTH levels of MSP animals were higher than MLP and SSP animals, and that animals placed on the multiple platform tanks showed the highest ACTH levels on Day 4 of manipulation. CORT levels were elevated in the animals kept over small platforms, and these levels where higher on Day 1 than basal and further elevated on Day 4 of PS deprivation. These results indicate that the multiple platform technique induces a distinct activation of the HPA axis, and that PS deprivation may act as an additional stressor.