role of lactobacillus acidophilus loaded floating beads in chronic fatigue syndrome: behavioral and...

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

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.

Volume 24, Number 4, April 2012 LAB loaded FBs attenuate chronic fatigue syndrome

� 2012 Blackwell Publishing Ltd 367

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

P. K. Singh et al. Neurogastroenterology and Motility


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).



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.



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

A

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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.

Group I

60

a

a

a,b

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50

40

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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.



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

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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)


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



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.



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



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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