role of lactobacillus acidophilus loaded floating beads in chronic fatigue syndrome: behavioral and...
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Role of Lactobacillus acidophilus loaded floating beads in
chronic fatigue syndrome: behavioral and biochemical
evidences
P. K. SINGH, K. CHOPRA, A. KUHAD & I. P. KAUR
University Institute of Pharmaceutical Sciences, UGC Centre of Advanced Study, Panjab University, Chandigarh 160014, India
Abstract
Background In recent years the interface between
neuropsychiatry and gastroenterology has converged
in to a new discipline referred to as enteric neurosci-
ence. Implications of brain–gut communication in the
pathogenesis of psychiatric disorders indicate a pos-
sible role of suitably packaged/delivered probiotics as
newer therapeutic options. In the present study prob-
able role of per-oral administration of free Lactoba-
cillus acidophilus (LAB) and LAB loaded alginate
beads in attenuation of the symptoms associated with
chronic fatigue syndrome (CFS) were evaluated.
Methods Chronic fatigue syndrome following physical
fatigue was induced in rats by forcing them to swim
(forced swim test; FST) in water till exhaustion, after
weighing them down with 10% their body weight,
daily for 28 days. Immobility (I) and postswim fatigue
time (PSF) were taken as suitable markers. Free LAB
and LAB loaded floating beads (FBs) were adminis-
tered, from 21 to 28 days. Key Results Immobility and
PSF were found to increase considerably in FST rats
(665 ± 22 s and 196 ± 6 s) as compared with the naıve
(32 ± 7 s and 22 ± 2 s) at 20 days, establishing severe
fatigue like behavior. FST control group exhibited
significant (P < 0.05) hypertrophy of spleen, hypotro-
phy of thymus, and increased oxido-nitrosative stress
in brain and tumor necrosis factor-a (TNF-a) levels in
serum. Treatment with LAB and LAB FBs significantly
decreased I and PSF and attenuated (P < 0.05) oxido-
nitrosative stress and TNF-a levels. Spleen and thymus
were also restored to their original size in this group.
Conclusions & Inferences The findings suggest a
valuable therapeutic role of LAB especially when
incorporated into alginate beads for the treatment of
CFS.
Keywords chronic fatigue syndrome, forced swim
test, oxido-nitrosative stress, probiotic loaded
alginate beads, therapeutic option, TNF-a.
INTRODUCTION
In recent years the interface between neuropsychiatry
and gastroenterology has converged into a new disci-
pline referred to as enteric neuroscience. Emerging
studies have shown that intestinal bacteria can directly
communicate with the central nervous system (CNS)
through the vagal sensory nerve fibers and the periph-
eral immune system.1 The existence of a mutual
interaction between the brain and the gastrointestinal
(GI) system, organs that are functionally very different,
though seems unlikely, is supported in literature,
which presents compelling evidence for the existence
of a ‘brain–gut axis.’ It has been suggested that not only
can the brain affect gut function, but that the gut, by
both direct and indirect mechanisms, can also induce
changes in the CNS.2,3 This bidirectional communica-
tion is particularly relevant during stress4 and in stress-
related disorders such as depression.5 Orally consumed
lactic acid bacteria are reported to be useful in allevi-
ating depression like symptoms. Patients with mental
health disorders appeared to have very low levels of
Lactobacillus acidophilus (LAB) and in a series of case
reports in 1924, an Illinois physician reported that oral
LAB improves complexion in mental health disorders,
it was stated that ‘in certain patients it even seemingly
contributes to mental improvement.’6
Chronic fatigue syndrome (CFS) is a depressive and
incapacitating illness defined by disabling chronic
Address for Correspondence
Dr. Indu Pal Kaur, Professor of PharmaceuticsUniversity Institute of Pharmaceutical Sciences,UGC Centre of Advanced Study, Panjab University,Chandigarh 160 014, India.Tel: +91 172 2534191; fax: +91 172 2541142;e-mail: [email protected]: 2 July 2011Accepted for publication: 14 December 2011
Neurogastroenterol Motil (2012) 24, 366–e170 doi: 10.1111/j.1365-2982.2011.01861.x
� 2012 Blackwell Publishing Ltd366
Neurogastroenterology & Motility
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fatigue and characteristic accompanying signs.7,8
Researchers have documented lower levels of bifido-
bacterium and higher levels of enterococcus species in
these patients. Disturbances in intestinal microbial
ecology and in the immune system of the host have
been implicated as a part of the pathogenesis in CFS.
Probiotic lactic acid producing bacteria have been
shown to prevent and alleviate GI disturbances and to
normalize the cytokine profile which might be of an
advantage for patients suffering from CFS. Among
factors that have been implicated in triggering or
mediating the course of CFS are infective disorders,
immune system disorders, neuroendocrine abnormal-
ities and neuropsychological impairment.9,10 It has
been suggested that immune dysfunctions observed in
CFS patients account for a number of the described
symptoms.11 The CFS patients have a cytokine
imbalance in the peripheral blood compartment and
the system is biased towards a T-helper (Th) type 2
immunity-oriented pattern. Specific strains of probi-
otics may alter cytokine secretion and act as potent
antioxidants within and beyond the GI tract
(GIT).12,13
There is evidence that the activation of immune
inflammatory process and abnormalities in lipids may
cause overproduction of reactive oxygen species (ROS),
lipid peroxidation and antioxidant enzyme activities
and these processes may be related to the pathophys-
iology of depression or depression like symptoms.14
Many depression patients showed increased levels of
proinflammatory cytokines like interleukin (IL)-1 and
tumor necrosis factor-a (TNF-a).15,16
To achieve the claimed benefits, probiotics have to
reach their site of action in sufficient numbers. Studies
have shown that numbers of viable probiotic cells tend
to decline both in probiotic supplemented food prod-
ucts17–21 and during GI transit.22–24 This has resulted
in an ongoing search for methods that will improve
probiotic viability in both these cases. Microencapsu-
lation is one of the techniques employed to protect
bacteria against adverse conditions during manufacture
and storage of the product and its transit in the GIT.
Microencapsulation refers to a technology of packaging
solids, liquid, or gases in microscopic sealed capsules
that can release their contents at controlled rates under
the influence of specified conditions.25,26 Several stud-
ies have demonstrated successful entrapment of living
microbial cells within various semi-permeable poly-
meric gel structures.22,27–30 We have also reported on
the entrapment of LAB within alginate beads to
prolong their stay in GIT (for better establishment in
gut mucosa), maintaining the viability especially in
acidic conditions of the stomach. The developed beads
also showed an enhanced performance in the gastric
ulcer model.31,32
Objective of this study was to assess free LAB and its
formulated beads in suitable animal model of CFS
using the forced swim test (FST)33 to induce chronic
fatigue. Tanaka et al. established an animal model for
combined (mental and physical) fatigue using a weight
loaded forced swimming test which leads to oxidative
stress, and the latter is also involved in the pathogen-
esis of CFS.34,35 Oxidative stress affects both physical
and mental function through various redox-sensitive
signaling systems.34 As the biological effects of Lacto-
bacillus are initiated at the level of the gut, whereas
pathological abnormalities of CFS/depressive illness
predominantly originate in the brain, any potential
effects of this treatment are likely to be mediated via
the two major interacting pathways that comprise the
oxido-nitrosative stress and immune system. There-
fore, oxido-nitrosative stress markers were measured in
brain homogenates of CFS induced and treatment
animals. Furthermore, the expression of inflammatory
cytokine TNFa was determined in rat serum as a
cytokine imbalance is reported in the peripheral blood
compartment of CFS patients36–38 and forms a more
suitable and clinically relevant marker than its deter-
mination in brain homogenates. Further the immobil-
ity (I) and PSF times were also noted post-FST for
3 weeks (21 days) and treatment accompanied by FST
for the 4th week. This time has been observed to result
in peak and a chronic fatigue (lesser periods of time
may show an acute response). Effects obtained with
free LAB were compared with LAB FBs to determine
appropriateness of the developed microencapsulating
system. The study is a first report on the use of LAB in
an animal model of CFS. A few clinical studies are
reported with some lactobacillus and bifidobacterium
species but they involved long-term administration of
probiotics (‡4 weeks) and did not indicate significant
change in fatigue and physical activity scores.37
MATERIALS AND METHODS
All the chemicals or reagents used in the study were of analyticalreagent or guaranteed reagent grade.
Probiotic and its dosing
Probiotic (LAB) was procured as a gift sample from RanbaxyGurgaon, India [not less than 200 billion Colony forming unit(CFU) gm)1]. Lactobacillus acidophilus or LAB FBs were dis-persed in sterile distilled water containing 1% carboxy methylcellulose and treatment was started postinduction of CFS byexposing to FST for up to 20 days. 107 CFU dose of LAB or its FBsequivalent to 107 CFU were administered by oral gavage, from21st to 28th days, 30 min prior to the FST.
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Preparation of LAB loaded calcium alginate FBs
Calcium alginate beads were prepared by orifice ionic gelationmethod. Measured amount (20 mg LAB) of the probiotic wassuspended in water. Details of the procedure are reportedearlier.31,39,40 The beads were prepared aseptically using sterilesolutions, vehicle and container.
Viability study of probiotic loaded floating beadsin simulated gastric fluids (SGF)
Viability of probiotic entrapped within the beads was carriedout in SGF under aseptic conditions. Measured amount ofbeads/free probiotic was incubated anaerobically at 37 �C intest tubes containing 10 mL of SGF. At intervals of 2, 4, and6 h of incubation, respectively, all the beads from tubes ateach time point, were removed, washed with peptone waterand immediately assayed for cell count, Tubes containing freeprobiotic were centrifuged at respective time points and pelletsfrom each tube were used for cell enumeration after suitabledilution.
K ¼ log aða�xÞt Where K represents rate constant of death/kill,
a initial number of bacteria in the medium and ‘(a)x)’ the numberof organisms in the same volume after exposure for time t.
Animals used
Adult male Wistar rats (250–270 g) bred in the Central AnimalHouse facility of Panjab University, Chandigarh, India wereused for the study. The animals had free access to standardrodent food pellets and water. They were acclimatized to thelaboratory conditions before the experiment. All the experi-ments were conducted between 09 : 00 h and 17 : 00 h. Theexperimental protocols were approved by the InstitutionalAnimal Ethics Committee (IAEC) of Panjab University,Chandigarh (Reference No. 972/DUI/PU; Dated February 08,2010).
EXPERIMENTAL PROCEDURES
The animals were divided into six groups, consisting
of five to six animals in each group. Group I (Naive
control) consisted of animals subjected to swim only
on first day. Group II (per se) consisted of control
animals receiving LAB FBs (107 CFU) from 21st to
28th day. Animals of this group were exposed to FST
only once a week, i.e., on day 1, 7, 14, 20, 21, and 28
of the study and the values thus obtained were used
as controls for comparison with the CFS and treat-
ment groups (III–VI). Group III (CFS control group)
comprised of animals subjected to weight loaded
forced swimming for 28 days, Group IV, V, and VI
were similarly subjected to weight loaded forced
swimming for 28 days with suitable treatments for
the last week (21–28 days) as blank beads (placebo
group; group IV); LAB 107 CFU (free probiotic group;
group V) and LAB FBs equivalent to 107 CFU (group
VI), respectively.
Induction of CFS and depression like symptomsby forced swim test
Rats bearing a load of 10 ± 2% of their body weight
were plunged individually and forced to swim inside a
rectangular glass jar (60 · 30 · 45 cm3) containing
30 cm3 of water maintained at 22 ± 3 �C; the depth of
the water was adequate to prevent the animals from
touching the bottom of the floor with their tails.33 The
animals were forced to swim in the jar till exhaustion
daily for 28 days.
Behavioral assessment
After an initial period of vigorous activity, each animal
assumed a typical immobile posture by barely keeping
themselves afloat and their nose clear of water. The
rats were considered to be immobile when they ceased
to struggle and made minimal limb movements, to
keep their head above the water level. This was taken
as the ‘immobility period’ and was followed by com-
plete exhaustion, which was defined as the point at
which the rat’s noses remained below the water surface
for up to 10 s.41 When signs of marked fatigue/
exhaustion became apparent, the animals were
removed from the water and placed in a clear observa-
tion chamber. The time elapsed before rat initiated
grooming (licking and rubbing of the skin/fur) was
recorded to assess the ‘postswim fatigue’ (PSF). Latter
is the time taken by the animals before they start
grooming and the same was recorded in seconds after
each rat was removed from water.42,43
Blood collection, animal sacrifice, and tissuedetection
On the 29th day blood was collected through tail vein
of animals, after which the animals were sacrificed to
harvest their brain, spleen, and thymus. Brains (placed
in cold 0.1 mol L)1 phosphate buffered saline) were
weighted and stored at )80 �C until the preparation of
homogenates.
Preparation of brain homogenates andoxido-nitrosative stress determinations
A 10% w/v brain homogenate was prepared in
0.1 mol L)1 phosphate buffer (pH 7.4) saline which
was further used for lipid peroxidation,44 catalase
(CAT),45 superoxide dismutase assay (SOD),46 protein
estimation,47 and measurement of nitrosative
stress.48
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TNF-a estimations
The quantification of TNF-a was done in serum as per
the instructions provided by research and development
(R&D) Systems Quantikine rat TNF-a immunoassay
kit. The enzyme reaction yields a blue product that
turns yellow when the stop solution is added. Intensity
of the measured color is in proportion to the amount of
rat TNF-a bound in the initial steps. The sample values
are then read off the standard curve.
Correlation between induced behavioral changes,biochemical changes, and TNF-a level
The extent of induced behavioral changes (I and PSF) in
various groups was plotted against the corresponding
change in the various biochemical parameters (Nitrite,
LPO, SOD, and CAT) as observed on the 28th day of the
study to evaluate whether the induced fatigue, as
expressed by change in I and PSF, is also manifested in
terms of oxido-nitrosative stress. Similar plot was made
betweeninducedbehavioralchanges(IandPSF)andTNF-
a level.
Statistical analysis
Raw data obtained from viability studies are expressed as
mean ± SD. The results for I and PSF in the forced swim
exercise were measured in seconds and expressed as
mean ± SEM. The intergroup variation in behavioral and
biochemical tests was measured by one-way analysis of
variance (ANOVA) followed by post hoc test (Bonferonni).
Statistical significance was considered at P < 0.05.
RESULTS
Viability study of probiotic loaded in to FBs in toSGF and its transit through GIT
Results (Fig. 1) show a decrease in bacterial count after
exposure of free probiotic or probiotic beads to SGF at
37 �C at different time points. However, the rate
constant of death/kill (K) at all time points was
significantly less for the probiotic loaded in to beads
as compared with the free probiotics (Table 1). This
confirmed that encapsulation of probiotic bacteria
within FBs protected them from the harsh acidic
conditions in gastric fluids. Results show the superi-
ority of the FBs system for maintaining the viability of
LAB in gastric environment. Earlier studies with
similarly prepared FBs show an extended stay of the
beads in GIT32 allowing greater time for the slowly
released probiotic to establish in the gastric mucosa.
Preliminary studies to establish the onset of CFS,as indicated by I and PSF
Animals were exposed to FST everyday and I and PSF
were determined at regular weekly intervals (Table 2).
The 3 week period was considered suitable as periods
£2 weeks may only manifest acute fatigue (which may
be reversible). However, earlier studies by our group
showed that 3 weeks of exposure to FST induced
symptoms which were clinically relevant to
CFS.42,43,49 Periods exceeding 4 weeks resulted in an
extreme fatigue when rats made no effort to keep
themselves afloat and started to drown as soon as they
were put in to water. Treatment with LAB and its
loaded FBs were thus initiated from day 21 (postinduc-
tion of CFS) and continued over a week up to the 28th
day from the start of FST.
Effect of LAB and LAB FBs on I and PSF
The FST significantly increased I (Table 2) and PSF in
rats (665 ± 22 s and 197 ± 6 s, respectively) as com-
pared to the naive control group (32 ± 7 s and 22 ± 2 s,
respectively) or the per se group (40 ± 5 and 32 ± 3,
respectively) at 20 days, indicating the onset of CFS. At
28 days, I was 1140 ± 38 s and PSF was 273 ± 7 s for
the FST control rats (Group III). Oral administration of
LAB from 21st day onwards significantly (P < 0.05)
decreased I as well as PSF time as compared with the
Table 1 Rate constant of death/kill of free probiotic and probiotic
loaded FBs in SGF (n = 4)
Time (h)
K value
Free probiotic Probiotic floating beads
2 )0.1135 )0.2147
4 0.1072 )0.0857
6 0.3766 0.0033
All values are significantly (P < 0.05) different at each time points.
100Probiotic beads Free probiotic
90
80
70
60
50
% S
urvi
val
40
30
20
10
00 2
Time (h)4 6
Figure 1 % Survival of bacteria after exposure to simulated gastric
fluids at 37 �C at different time intervals (n = 4).
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FST control group. LAB FBs showed a significantly
enhanced reducing effect on these parameters in
comparison with the LAB treated group. Administra-
tion of LAB FBs showed a significant effect on I and PSF
as compared with the FST group [F(6, 29) = 354.980
(P < 0.001); F(6, 29) = 333.730 (P < 0.001)].
Effect of LAB and LAB FBs on spleen and thymusgland
The 28 days forced swimming fatigue, led to a signif-
icant hypertrophy of spleen and hypotrophy of thymus
gland in FST control rats as shown in Table 3. Treat-
ment with LAB FBs showed a significant (P < 0.05)
reduction in induced hypertrophy of spleen. The
achieved effect was significantly better than that
achieved with free LAB group. LAB and LAB FBs also
showed significant improvement in the induced hypo-
trophy of thymus in FST control group with LAB FBs
showing arithmetically better results. LAB FBs admin-
istered group show significant effect on weight of
spleen and thymus as compared with the FST group
[F(6, 29) = 49.039 (P < 0.001); F(6, 29) = 10.171 (P < 0.001)].
Effect of LAB and LAB FBs on body weight
The FST control group (Group III) showed a significant
reduction in the overall body weight. Administration
of LAB and its FBs, however, showed improvement in
the body weight of treated rats as shown in Table 3.
LAB FBs administered group show significant effect on
body weight as compared with the FST group
[F(6, 29) = 632.362 (P < 0.001].
Effect of LAB and LAB FBs on oxido-nitrosativestress
Malonaldehyde (MDA) and nitrite levels were signifi-
cantly increased in the brain of FST rats as compared
with the naive control group. Treatment with LAB FBs
not only produced a significant reduction (P < 0.05) in
nitrite level, but the effects were more significant than
the free LAB group (Fig. 2A) and completely reversed
the induced changes. In case of MDA level LAB FBs
showed arithmetically more reduction of MDA as
compared with the free LAB and also approached the
level of naive control but it was not significantly
different at P < 0.05 (Fig. 2B). LAB FBs administered
group show significant effect on MDA and nitrite
levels in rat brain as compared with the FST group
[F(6, 29) = 25.984 (P < 0.001); F(6, 29) = 51.325 (P < 0.001)].
Results clearly establish the therapeutic use of LAB
FBs in chronic fatigue induced stress. Enzyme activity
of superoxide dismutase and CAT was significantly
decreased in the brains of CFS rats as compared with
naive control. Treatment with LAB FBs produced
significant (P < 0.05) increase in CAT level; and the
effects were better (P < 0.05) than free probiotic treated
Table 3 Effect of LAB and LAB FBs on %change in body weight,
thymus/body weight ratio, and spleen/body weight ratio in load
induced-chronic fatigue (FST) in rats
Groups
Thymus/body
weight (mg g)1)
Spleen/body
weight (mg g)1)
%Change in
body weight
Group I 0.33 ± 0.045 0.56 ± 0.028 8.88 ± 3.63
Group II 0.36 ± 0.042 0.52 ± 0.12 8.19 ± 1.93
Group III 0.13 ± 0.021* 1.65 ± 0.089* )14.64 ± 2.24*
Group IV 0.14 ± 0.028* 1.60 ± 0.068* )15.22 ± 3.67*
Group V 0.22 ± 0.020* 1.04 ± 0.065*,� )11.78 ± 2.08*
Group VI 0.27 ± 0.013� 0.69 ± 0.029�,� )9.40 ± 2.53*
Group I: Naive control; Group II: per se LAB FBs (equivalent to
107 CFU); Group III: FST; Group IV: FST induced depression group
receiving blank beads; Group V: FST group receiving LAB (107 CFU);
Group VI: FST group receiving LAB FBs (equivalent to 107 CFU). LAB,
Lactobacillus acidophilus; FBs, floating beads.
*P < 0.05 as compared with group I and group II, �P < 0.05 as compared
with group III and IV, �P < 0.05 as compared with group V.
Table 2 Effect of LAB and LAB FBs on immobility (s) and postswim fatigue (s)
Group
1 Day 7 Day 14 Day 20 Day 21 Day 28 Day
I PSF I PSF I PSF I PSF I PSF I PSF
Group I* 32 ± 7 22 ± 2
Group II 33 ± 11 28 ± 5 45 ± 11 28 ± 4 45 ± 6 30 ± 5 40 ± 5 32 ± 3 46 ± 4 33 ± 2 53 ± 4 32 ± 2
Group III 43 ± 10 33 ± 4 125 ± 13� 91 ± 7� 287 ± 21� 160 ± 8� 665 ± 22� 197 ± 6� 773 ± 19� 204 ± 6� 1140 ± 38� 273 ± 7�
Group IV 40 ± 16 26 ± 5 97 ± 4� 52 ± 5� 271 ± 24� 96 ± 5� 679 ± 30� 177 ± 7� 808 ± 37� 185 ± 7� 1132 ± 46� 265 ± 6�
Group V 32 ± 5 29 ± 5 102 ± 5� 51 ± 6� 279 ± 24� 103 ± 6� 656 ± 14� 185 ± 7� 636 ± 13�,� 180 ± 7� 529 ± 7�,� 156 ± 8�,�
Group VI 40 ± 8 27 ± 5 90 ± 7� 58 ± 6� 276 ± 12� 115 ± 7� 679 ± 31� 209 ± 11� 619 ± 21�,� 199 ± 12� 384 ± 22�,�,§ 92 ± 8�,�,§
Group I: Naive control; Group II: per se LAB FBs (equivalent to 107 CFU); Group III: FST; Group IV: FST induced CFS group receiving blank beads;
Group V: FST group receiving LAB (107 CFU); Group VI: FST group receiving LAB FBs (equivalent to 107 CFU). I, immobility; PSF, postswim fatigue;
LAB, Lactobacillus acidophilus; FBs, floating beads.
*Naive control subjected to FST on 1st day.�P < 0.05 as compared with group I and group II, �P < 0.05 as compared with group III and IV, §P < 0.05 as compared with group V.
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group (Fig. 2C) In case of SOD level LAB FBs show
arithmetically more increase of SOD as compared with
the free LAB approaching the level of naive control but
it was not significant at P < 0.05 (Fig. 2D). LAB FBs
administered group show significant effect on SOD and
CAT levels in rat brain as compared with the FST group
[F(6, 29) = 29.150 (P < 0.001); F(6, 29) = 77.316 (P < 0.001)].
Measurement of serum TNF-a
Serum TNF-a level was markedly increased in CFS
induced rats. It is suggested that swim stress increases
the permeability of the blood brain barrier (BBB)50 thus
TNF-a in the brain reaches into the blood after crossing
the BBB. Researchers show that serum TNF-a level is
increased in chronic fatigue patients and is consistent
with the findings in the in vitro production of cytokines
from monocytes of patients with CFS.51–53 This obser-
vation indicates a probable role of proinflammatory
cytokines in this disease. LAB produced significant
decrease (P < 0.05) in TNF-a as compared with CFS
group. LAB loaded beads showed significantly better
reduction in TNF-a approaching the level of naive rats
(Fig. 3). LAB FBs administered group show significant
effect on TNF-a levels in serum of rat as compared with
the FST group [F(6, 29) = 33.373 (P < 0.001)].
Correlation between induced behavioral changes,biochemical changes, and TNF-a level
Figs 4 and 5 show that significant correlation exists
between the oxido-nitrosative stress and the behavioral
response of increased I and PSF in the chronically
fatigued and the treatment groups, establishing that
chronic fatigue led to oxido-nitrosative stress and vice
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Group I Group II Group III Group IV Group V Group VI Group I Group II Group III Group IV Group V Group VI
Group I Group II Group III Group IV Group V Group VIGroup I Group II Group III Group IV Group V Group VI
Figure 2 Effect of LAB and LAB FBs on nitrite levels (A), lipid peroxides (MDA) (B), catalase levels (C), and superoxide dismutase (D) in weight loaded
water immersion stress model of depression in rats. Group I: Naive control; Group II: per se LAB FBs (equivalent to 107 CFU); Group III: FST; Group
IV: FST induced depression group receiving blank beads; Group V: FST group receiving LAB (107 CFU); Group VI: FST group receiving LAB FBs
(equivalent to 107 CFU). aP < 0.05 as compared with Group I and Group II, bP < 0.05 as compared with group III and IV, cP < 0.05 as compared with
Group V.
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Figure 3 Effect of LAB and LAB FBs on serum TNF-a level in CFS rats.
Group I: Naive control; Group II: per se LAB FBs (equivalent to
107 CFU); Group III: FST; Group IV: FST induced depression group
receiving blank beads; Group V: FST group receiving LAB (107 CFU);
Group VI: FST group receiving LAB FBs (equivalent to 107 CFU).aP < 0.05 as compared with group I and group II, bP < 0.05 as compared
with group III and IV, cP < 0.05 as compared with group V.
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versa. MDA, nitrite and TNF-a show a positive corre-
lation to increase in I and PSF which is also proportional
to a corresponding decrease in SOD and CAT levels.
DISCUSSION
Present investigation is a preliminary study taken up
to evaluate the proposed concept of enteric neurosci-
ence and to establish the neurobiological role of
probiotics, especially if they are suitably formulated
to improve their viability and stay in the GIT. Such
effects of probiotics are not documented in preclinical/
animal model of CFS. To our knowledge there is only
one report in rats which indicates antidepressant
effects of Bifidobacterium.1 Animals were exposed
chronically (28 days) to FST in the present investiga-
tion so as to induce CFS. FST is based on the
observation that the animal develops an immobile
A B C
FED
16.0014.0012.0010.00
8.00
mol
es o
f MD
A/m
g pr
otei
n
6.004.002.000.00
16.00 30.00
25.00
20.00
15.00
10.00
5.00
0.00
14.0012.0010.008.00
mol
es o
f MD
A/m
g pr
otei
n
n m
oles
/mg
prot
ein
30.00 60.00
50.00
40.00
30.00
20.00
10.00
0.00
25.00
20.00
15.00
10.00
5.00
0.00
n m
oles
/mg
prot
ein
Con
c. (p
g/m
l)
60.00
50.00
40.00
30.00
20.00
10.00
0.00
Con
c. (p
g/m
l)
6.004.002.000.00
0.00 50.00 100.00 150.00 200.00 250.00 300.000 200
y = 0.008x + 4.66R2 = 0.935
y = 0.082x + 4.474R2 = 0.995
y = 0.020x + 21.87R2 = 0.926 y = 0.093x + 20.04
R2 = 0.947
y = 0.036x + 3.938R2 = 0.959 y = 0.018x + 6.066
R2 = 0.978
400
Immobility (s) Post swim fatigue (s)
0.00 50.00 100.00 150.00 200.00 250.00 300.00Post swim fatigue (s)
0.00 50.00 100.00 150.00 200.00 250.00 300.00Post swim fatigue (s)
Immobility vs MDA Immobility vs Nitrite
Immobility vs TNF-α
PSF vs MDA
PSF vs Nitrite PSF vs TNF-α
600 800 1000 1200
0 200 400Immobility (s)
600 800 1000 1200
0 200 400
Immobility (s)
600 800 1000 1200
Figure 4 Correlation between induced behavioral changes with biochemical (MDA and nitrite) and TNF-a. Immobility (I) vs lipid peroxides
(MDA) (A), postswim fatigue (PSF) vs lipid peroxides (MDA) (B), I vs nitrite (C), PSF vs nitrite (D), I vs TNF-a (E), PSF vs TNF-a (F). Above values are taken
at 28th day to correlate behavioral changes (I and PSF) with corresponding biochemical changes (LPO and nitrite) and TNF-a level for all the groups.
A B
C D
18.0016.0014.0012.0010.00
8.00
SO
D U
nits
/mg
prot
ein
6.004.002.000.00
18.0016.0014.0012.0010.008.00
SO
D U
nits
/mg
prot
ein
6.004.002.000.00
0 200 400 600 800 1000 1200
y = –0.011x + 14.01R2 = 0.845
y = –0.015x + 23.28R2 = 0.948 y = –0.071x + 24.65
R2 = 0.962
y = –0.053x + 15.05R2 = 0.866
Immobility (s)
0 200 400 600 800 1000 1200
Immobility (s)
Immobility vs SOD
Immobility vs Catalase PSF vs Catalase
PSF vs SOD
0.00 50.00 100.00 150.00 200.00 250.00 300.00Post swim fatigue (s)
0.00 50.00 100.00 150.00 200.00 250.00 300.00Post swim fatigue (s)
30.00
25.00
20.00
15.00
10.00
5.00
0.00
30.00
25.00
20.00
15.00
10.00
5.00
0.00
μ m
oles
of H
2O2 d
ecom
pose
/mg
prot
ein
μ m
oles
of H
2O2 d
ecom
pose
/mg
prot
ein
Figure 5 Correlation between induced behavioral changes with biochemical [superoxide dismutase (SOD) and catalase (CAT)] immobility (I) vs
SOD (A), postswim fatigue (PSF) vs SOD (B), I vs CAT (C), PSF vs CAT (D). Above values are taken at 28th day to correlate behavioral changes
(I and PSF) with corresponding biochemical changes (SOD and CAT) for all the groups.
P. K. Singh et al. Neurogastroenterology and Motility
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posture when placed in an inescapable cylinder filled
with water. In this paradigm, I is interpreted as a
passive stress coping strategy (behavioral despair).
Exposure of animals to a chronic FST indicates an
onset of symptoms similar to CFS as indicated by a
significant increase in I and PSF time.
We evaluated the effect of administering LAB to rats
with CFS and observed a significant improvement
which was further enhanced by incorporating LAB into
FBs. Latter improves the stay of LAB with in stomach
to more than 10 h31,32,40 while in normal routine orally
administered material passes out of the stomach
within 2–4 h. Further to this, incorporation within
FBs improves the viability of LAB in the gastric fluid as
shown in Table 1.
Several studies focus on the involvement of ROS in
the pathophysiology of fatigue and CFS.54–56 CNS is
extremely sensitive to free radical damage because of
its relatively small total antioxidant capacity.57 Theo-
retically, oxidative stress can be induced by an increase
in the generation of ROS, or by a decline in the
efficiency of antioxidant enzyme systems. Evidence of
oxidative damage to the deoxyribonucleic acid and
lipids of biopsy samples from the vastus lateralis
muscles of psychiatric patients has been observed.58
Oxidative stress once induced is not expected to be
limited to a specific area of the brain. The literature is
replete with evidences where whole brain homogen-
ates are used for these determinations.59,60 The use of
specific brain parts implicated in CFS can, however,
result in more significant studies.
We observe a significant oxido-nitrosative stress in
the presented model of modified FST (CFS) (Fig. 2).
However, treatment with LAB and LAB FBs signifi-
cantly attenuated the induced stress, with LAB FBs
treatment being significantly better. It was possible to
completely reverse the induced elevation of MDA and
nitrite levels by LAB FBs as indicated by no significant
difference from naive control and per se values post-
treatment with LAB FBs for 1 week. Similarly the
compromised antioxidant enzyme (SOD and CAT)
defense system(s) were also restored by the adminis-
tration of LAB and LAB FBs (Fig. 2). Furthermore, the
induced oxido-nitrosative stress correlates signifi-
cantly (R2 > 0.9) with the increase in I and PSF for
CFS induced animals (Fig. 4) thus confirming the
involvement of ROS in pathophysiology of CFS.
Decrease in SOD levels may be due to its utilization
in combating the increased production of O2·) subse-
quent to fatigue induced by the chronic forced swim-
ming regime suffered by the animals. Further,
hydrogen peroxide has been implicated in various
stress conditions and the former is decomposed by
CAT. Significantly (P < 0.05) decreased CAT activities
were observed in the brain of CFS group as compared
with the naive control group, indirectly indicating high
H2O2 production, such that more of CAT is being
utilized in the process. That is why the induced CFS (in
terms of I and PSF) shows a negative correlate with
SOD and CAT levels. It may thus be concluded that
the biochemical changes observed for various groups in
brain homogenates are due to the induced CFS as
shown by their significant correlation with behavioral
responses. Similar observation can be made for TNF-a.
It is now well-established that there are complex
bidirectional interactions between the CNS and the
immune system.61 During an immune response, an
important reciprocal relationship between immune
products and brain function occurs and this cross-talk
is essential to maintain homeostasis.62 Thymus is an
organ which is easily shrunk or reduced by physical,
mental, and emotional stress. A significant hypotrophy
of thymus was observed in rats exposed to FST daily for
20 and 28 days, respectively (Table 2).
Spleen is an association between physical lassitude
and immunity suppression. Various studies have
reported that increase of spleen weight indicates
alteration in the immune function.42 Spleen trans-
forms and transports the nutrients and fluids of the
body and dominates the muscle; if spleen is affected
then muscle weakness, arthritic symptoms, or swell-
ing of the joints may ensue.63 So an altered spleen
weight observed in the present study in CFS control
group may be associated with fatigue and associated
symptoms in animals.
Literature indicates that LAB have the potential to
influence the immune system in depressed patients by
supporting Th1 in driven cellular immunity and may
also decrease allergies. In addition, LAB are strong
antioxidants (in vitro and in vivo test systems),64 may
improve essential fatty acids status, can enhance
absorption of micronutrients by protecting the intesti-
nal epithelial barrier, and have been used to treat small
intestinal bowel diseases.65,66 Effect of probiotics on
the feel-good neurotransmitter L-tryptophan is also
documented.67 It has been reported that bacteria in the
GIT can communicate with the CNS and influence
behavior associated with emotion, anxiety, and major
depressive disorder, even in the extremely low
level and in the absence of an immune response.68,69
A significant improvement in behavioral response
(I and PSF) in rats exposed to FST regularly for 28 days;
on treatment with LAB reaffirm the above observa-
tions. Furthermore, a significantly better effect being
observed with LAB FBs indicate the possibility of a
better crosstalk being established by the gut microflora
Volume 24, Number 4, April 2012 LAB loaded FBs attenuate chronic fatigue syndrome
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with CNS transmitters attributable to a prolonged stay
of FBs in the gut.
Studies have reported the role of proinflammatory
cytokines during physical stress.70,71 A hypersecretion
of proinflammatory cytokines during stress caused by a
hypofunctional neuroendocrine counter regulation has
been indicated as a possible explanation of exercise- or
stress-induced exacerbation of fatigue experienced by
patients with depression.72 TNF-a is also known to
play an important role in depression as it results in
decreased food intake by a centrally mediated effect
leading to body weight loss.73 A study performed1 to
evaluate antidepressant properties of probiotic (Bifido-
bacteria infantis) in force swim test animal models
indicated the significant attenuation of IFN-c, TNF-aand IL-6 levels. Effect of LAB on proinflammatory
cytokines and the immune system is also reported.74,75
Our experimental results also indicate similar effects.
This is also indicated in Fig. 4 where in the value of
TNF-a increases for CFS group showing correspondingly
greater values for I and PSF (R2 > 0.9) also.
What, however, needs to be noted is the fact that
treatment with LAB FBs did not only produce a
significantly better effect than free LAB but also
completely reversed the induced changes in TNF-a so
as to match with those obtained for naive control
values clearly establishing the therapeutic potential of
the developed beads.
CONCLUSION
The results from the current study suggest the involve-
ment of oxido-nitrosative stress, proinflammatory
cytokines and immune function in various behavioral
and biochemical deficits associated with chronic FST
induced fatigue. Administration of LAB induced sig-
nificant antistress effects as indicated by attenuation of
oxido-nitrosative stress, TNF-a and induced behavioral
symptoms upon its oral administration. Lactobacillus
loaded FBs were able to attenuate and often completely
reverse the induced changes (effect being significantly
better than free LAB) possibly by virtue of a multitude
of effects as discussed above. Major among them is a
gut induced crosstalk with the CNS; latter being
ensured by a prolonged and continuous release of
viable LAB from LAB FBs in the stomach. Clinical
studies report an administration of almost 4 week of
probiotic to achieve some effects; while significant
change in physical fatigue was not observed.37 It may,
however, be highlighted here that administration of
LAB FBs for 1 week could completely attenuate all the
biochemical, behavioral and immune changes induced
in CFS rats. Based on this, LAB FBs may thus find a
place in the therapeutic armamentarium for the treat-
ment of patients with CFS. Study was especially
planned to evaluate the curative role of LAB FBs in
the treatment of CFS, with the treatment being
initiated only after the induction of severe symptoms
while most natural agents evaluated for CFS in rodents
involve a simultaneous or a pretreatment.
ACKNOWLEDGMENT
Authors are thankful to Ranbaxy Gurgaon, India for providing giftsample of Probiotic (Lactobacillus acidophilus). The authors arealso grateful to Ms Sushma for helping with microbiologicalstudies.
FUNDING
No funding declared.
DISCLOSURES
No competing interests declared.
AUTHOR CONTRIBUTIONS
IPK designed the study, analyzed data, wrote paper. PKS per-formed research, analyzed data, wrote paper. KC guided about CFSModel and cytokinines determination. AH helped to perform thecytokinines studies.
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