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To evaluate the molecular mechanisms of effects of exercise training on cardiovascular


and cognitive parameters in patients with Chronic Obstructive Pulmonary Disease

Reference No : 252017002323
Saved By : Anand Prakash Yadav
Saved Date : 29-Apr-2017

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Proposal

Proposal

Reference No : 252017002323

Proposal Details

Project Title : To evaluate the molecular mechanisms of effects of exercise training on cardiovascular and
cognitive parameters in patients with Chronic Obstructive Pulmonary Disease
Scheme : National Post Doctoral Fellowship (N-PDF)
Broad Area : Life Sciences
Name of Applicant : Anand Prakash Yadav Email ID : anandprakash83ster@gmail.com
Date of Birth : 03-Dec-1983 Contact No : 919718746695
Category : OBC Gender : Male
Designation : Research Associate Is differently abled : No
Department : Department of Physiology PI Address : House no.- 2645, 2nd floor, gali no. -
7, bihari colony, shahdara, delhi(ncr)
State : Delhi Pin Code : 110032
Name of the J.S. YADAV
Father/Spouse :

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Details of Post Doctorate

Details of Post Doctorate

Qualifying Degree : Phd


Degree Awarded? : Yes
Date of Degree Awarded : 22/09/2015
Qualifying Degree Thesis Title : Effect of Antarctic environment on Immune Response of Indian Expedition
Members
Subject : Life Sciences
Name of Research Supervisor/Guide : Dr. Lilly Ganju
Name of the awarding University/Institute Bharathiar University/ Defence Institute of Physiology and Allied sciences
:
Brief details of Thesis work :
During PhD, my main focus was to study the effect of Antarctic environment on the immune and biochemical responses of
the personnels participating in the Indian Antarctic expedition. For conducting the study I participated in 28th Indian
Antarctic expedition as a winter team member and stayed in Antarctica for 13 months. The study was the first of its kind to
reveal the effects of ship born journey in southern ocean on human immune system , it was also the first study, which
traces the overall impact of the Antarctic environment including ship journey and 11 months stay in Antarctica on
biochemical and metabolic profile of Indian Antarctic expeditioners. Investigations were carried out to observe the effect of
multiple stresses like ship borne journey, isolation, cold, Magnetic field and UV exposure on human immune and
biochemical response. To achieve aims and objective several immunological, Proteomic and Metabonomic parameters
were estimated in sera and saliva samples collected at different time points from two expedition teams, first team
comprised of summer members, which stayed in Antarctica for a brief period of 3 months and mostly stayed on ship while
the second team constituted the members of wintering team who stayed at Antarctic research base Maitri for a period of
13 Months. The pre-exposure blood and saliva was collected at Delhi before the expeditioner proceeded to Antarctica as
base line control from both the teams. For short term exposure study, blood and saliva was collected second time, on
board in southern ocean in January 2009 and third collection was performed after being off board at Maitri station in
Antarctica. For long term stress exposure study blood samples were collected subsequently in four different stages of the
expedition from winter expeditioners in the months of March, May, August and November, while saliva was collected every
month from March to November 2009.
For evaluating immunological responses various components of immune systems were studied viz cell mediated, humoral
and mucosal immunity. For assessment of cell mediated immunity different cytokine and chemokine levels were
estimated, such as TNF-, IFN-, TGF-, IL-4 and chemokine MIP-1 in sera samples. Results indicated that, the ship
borne journey induced increase in TNF- and IFN- levels. And chemokine MIP-1 was increased while TGF- was
decreased at all time points in comparison to baseline levels in short term exposed study. In long term exposure, during
the initial phase of expedition there was a very sharp increase in the levels of pro-inflammatory cytokines like TNF-, IFN-
and chemokine- MIP-1, however these levels got decreased significantly in the latter half of the expedition, suggesting
there by TH1 biased immunity in short exposure study and initial phase of long exposure study while immunosuppression
in the latter half of the expedition.
Further assessment of Humoral immunity of both terms was done by estimation of Immunoglobulin- IgA, IgG and IgM in
sera samples. Results showed that in the short term exposure study, the levels of IgA increased significantly during the
ship journey as well as one month stay at Maitri. In long term exposure study, IgA levels were found to be increased
significantly in comparison to control throughout the stay at Maitri.
Changes in Mucosal immunity were assessed by estimation of secretory IgA, IgG, IgM along with TGF- and Salivary
cortisol saliva samples. Results indicated that in the short term exposure study, the ship borne journey induced increased
SIgA levels. While in wintering team, SIgA was increased throughout the stay at Maitri in comparison to baseline level
however significant alternation were not achieved in case of SIgG and SIgM.

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Stress Markers such as Cortisol, Netrin-1, sHLA-G, and Catecholamine were also estimated in sera samples of both
teams. Levels of cortisol were found to be decreased during ship journey as well as during one month stay at Maitri in
summer team members. However in winter team members the levels of cortisol were found to be increased during the
peak wintering period. Further secretary HLA-G was found to be significantly increased throughout the stay in both the
teams, suggesting it as a possible stress biomarker.
Proteomic study included the study of changes in expression of different proteins by estimation of Heat shock proteins
(HSP) - 60, 70 and 90 and 2D Gel electrophoresis followed by MALDI TOF analysis in the sera samples of both the teams.
The level of HSP-60 was found to be significantly increased during entire expedition in both the teams, while HSP-70 was
increased significantly in the winter team. Further, 2D gel electrophoresis and MALDI TOF analysis showed altered level of
complement proteins C3. A more comprehensive analysis of changes occurring in Complement system was done by
estimation of C3, C4, C3a, C4a, C5a and Factor B in the sera samples indicated activation of complement system during
the ship journey as well as during the peak wintering period in winter team.
Analysis of changes in metabolic profile was performed by 1H NMR for both the teams. For biochemical analysis,
conventional biochemical test were performed. The results of biochemical tests suggest that stress affected liver and
kidney functioning. While analysing NMR data it was observed that stress caused by extended ship journey and
seasickness prompted the stimulation of gluconeogenic pathways during the ship journey in southern ocean. Further
during wintering phase changes in energy metabolism were observed in addition to stimulation of gluconeogenic pathway,
which could be understood as bodys adaptive mechanism to counter the effects of stress.
Presently I am working on development of exercise protocol to improve hypoxia tolerance which will be helpful for soldiers
deployed at high altitude. For this, Ischemic Preconditioning is being used as an intervention for improving hypoxia
tolerance.

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Proposed Research Details

Proposed Research Details

Project Summary :
Chronic Obstructive Pulmonary Disease (COPD) is a chronic respiratory disease which is characterized by airflow
limitation; its usually progressive and associated with an abnormal inflammatory response of the lung to noxious gases
and particles. COPD is one of the major causes for hospitalization, emergency department visits and health care utilization
and results in an economic and social burden that is both substantial and increasing. In India; median prevalence of COPD
is estimated to be around 5% in men and 2.7% in women.
Though COPD manifests mainly as a pulmonary disease, it has significant extra-pulmonary component and is associated
with several co-morbid conditions such as, gas-exchange inefficiency, increased invasive and non-invasive ventilator
requirements, activation of coagulation factors, reduced muscle strength, pulmonary hypertension, cognitive impairments
and cardiovascular disorders that contribute to the severity of disease and mortality. Due to these co-morbidities, COPD
patients are at increased risk for myocardial infarction, angina, osteoporosis, respiratory infection, bone fractures,
depression, diabetes, sleep disorders, anemia and glaucoma. Frequent oxygen desaturation in these patients also
contributes to severity of above-mentioned comorbidities.
Several cellular and molecular alterations have been described in skeletal and respiratory muscles of patients with COPD
that includes mitochondrial function abnormalities and systemic inflammation. Reactive oxygen species, either directly or
via the formation of lipid peroxidation products, play a role in enhancing inflammation through the activation of stress
kinases and redox-sensitive transcription factors in patients with COPD.
Management of COPD includes pharmacological and non-pharmacological interventions. Pulmonary rehabilitation is one
of the evidence based, non-pharmacological interventions, which includes exercise training, nutritional counseling and
patient education and has been shown to reduce dyspnea, increase exercise performance, and improve health-related
quality of life. Exercise training enhances both the metabolic and functional status of the human body and leads to
increase in the expression of a number of genes involved in enhancement of physical capacity. It has been shown that
regular Exercise training induces mitochondria biogenesis in skeletal muscles, leading to an increase in muscle
mitochondria density and improvement of physical performance.
Since COPD reduces functional capacity and leads to poor quality of life along with adversely affecting different body
systems, including brain and heart, this study aims to examine the effect of exercise training on domains related to
cognition and cardiac autonomic function and evaluate its molecular mechanism. Thus the study will provide an insight into
the basis for improvement in patients with COPD which is conferred by exercise training and is relatively safe and has no
side effects.

Keywords : Exercise training, COPD, cognitive impairments, cardiac autonomic


functioning,
Objective :
1.To assess cardiovascular and cognitive parameters in patients with COPD.
2.To provide exercise training to patients with COPD.
3.To assess the effect of exercise training on cardiovascular parameters in patients with COPD.
4.To assess the effect of exercise training on cognitive parameters in patients with COPD.
5.To evaluate the molecular mechanisms underlying changes in cardiovascular and cognitive parameters after exercise
training in patients with COPD.

Expected Output and Outcome of the proposal :


Exercise Training will be used as an additional non-pharmacological intervention for management of COPD patients along
with their usual prescribed medication in this study.

Analysis of beneficial effect of the intervention will be the outcome of the study, which will be determined by:-

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1.The changes occurring in the functional capacity of the patients after exercise training which will be assessed by
changes in six-minute walk distance and body composition analysis.
2.The improvement occurring in the cardiovascular functioning in the patients after exercise training as reflected by
changes in Heart rate variability and Heart rate recovery.
3.The improvement occurring in the cognitive impairments in the patients after exercise training as reflected by changes in
the cerebral blood flow, visual evoked potential and cognitive performance assessments
4.Changes occurring in the expression of genes related to the signal transduction and metabolic functioning after exercise
training, which will help to understand the molecular mechanisms of effect of exercise training in COPD patients.

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Mentor Details

Mentor Details

Name & Address of the Host Institution : Vallabhbhai Patel Chest Institute , Delhi University, North Campus, Vijay
Nagar Marg, New Delhi, New Delhi-110007
Name of the proposed Mentor : Dr. Vishal Bansal
Designation : Assistant Professor
Department : Department of Physiology
Email Address : drvishalbansal@hotmail.com
Contact No : 9810525900
Email Address of Registrar : jrvpci@gmail.com
No. Ph.D. scholars working : 1
No. Postdoctoral fellows working : 0

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Reference Details

Reference Details

1 . Dr. Lilly Ganju


Sc"G"
Head, Immunomodulation Division,
Defence Institute of Physiology and Allied Sciences (DIPAS), DRDO, Lucknow Road, Timarpur, New Delhi-110054,
lganju@rediffmail.com , 9810383160

2 . Dr. Prasanna K Reddy


Sc"F"
Head, Cardio-Respiratory Physiology,
Defence Institute of Physiology and Allied Sciences (DIPAS), DRDO, Lucknow Road, Timarpur, New Delhi-110054,
drprasannakreddy@hotmail.com, 9958000217

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Work Plan and methodology

The study will be conducted at V. P. Chest Institute after institutional ethical clearance.
Written, informed consent will be obtained from all subjects prior to induction in the study.
Subjects selection
25 patients diagnosed with moderate to severe chronic obstructive pulmonary disease will be
enrolled in the study.
Sample size calculation
In a previous studies which have shown decrease in heart rate variability, one of the
parameters, namely, SDNN (ms) (standard deviation of all the NN intervals; which represents
a global index of HRV that reflects the influence of parasympathetic and sympathetic system
on heart); a difference in mean of 30 with standard deviation of 9 between normal controls
and COPD patients. For detecting this change post-intervention our study, we will require21
patients. Accounting for drop-outs (20%), enrollment of n=25 will suffice for our study.
Formula used: N= 22 [Z1-+ Z1-]2 Calculated for two-tail significance:
[1- 2]2 N= Sample size
= Mean of squares of Standard Deviation of
Pre and Post
Inclusion criteria: Z1-= 1.96, Z1- = 1.282
1=years
1. Male subjects with age range between 35 to 65 Meanwho
of Prehave
intervention, 2= Mean of
quit smoking at least
intervention
eight weeks before inclusion into the study.
2. Patients with a clinical diagnosis of moderate to severe chronic obstructive pulmonary
disease.
3. All patients should be compliant to pharmacotherapy for their disease.
4. Subjects willing to undertake the study regimens.

Exclusion criteria:
1.Patients having co-morbidities likely to affect the study parameters like cardiovascular
disorders, metabolic and endocrinal disease.
2.Subject having acute infection in the month preceding induction into the study or during
the study period and patients requiring course of oral steroids during the course of study
shall be excluded from the study.
3.Physiological, musculoskeletal, neurological or psychological impairment impeding
training program.

Study Design
Pre-Test and Post-Test, prospective study.

Methodology
Baseline value of the following parameters will be documented from the medical records
before including the subjects into the study: Haemoglobin, Red Blood Cells Indices, Total
leukocyte count (TLC), Differential leukocyte count (DLC), Fasting blood sugar (FBS),
Glycosylated Haemoglobin (Hb A1c), Pulmonary function test (PFT).
Following this, different outcome parameters as detailed below will be measured at following
time points
Week 0: At inclusion into the study, to assess cardiovascular and cognitive responses.
Week 10: At end of exercise training program, to assess cardiovascular and cognitive
responses post-intervention.

Methodology
Outcome Parameters
1. Six Minute Walk Test (6MWT):
6MWT will be performed to assess functional capacity on a long, flat, straight, enclosed
corridor with a hard surface as per the ATS guidelines. The walking course will be 40 m
in length and both the ends of the track will be marked with cones. Standard instructions
and encouragement will be given to the patients.

2. Body Composition Analysis:


Body composition analysis will be done by bio-impedance method.

3. Cerebral blood Flow:


Cerebral blood flow will be measured by Transcranial Doppler.

4. Cardiovascular Parameters
a. Heart Rate Variability (HRV)
Heart rate variability assessment will be carried out as per the recommendations of the
Task Force of the European Society of Cardiology and the North American Society of
Pacing and Electrophysiology. ECG will be recorded using standard lead II and fed into a
bio-amplifier and digitized using an analog-to-digital converter and then displayed on a
computer with the data acquisition and analyzing software. Regions of artifact will be
eliminated.
b. Heart Rate Recovery (HRR)
A graded leg ergometry will be performed in which all the subjects will be subjected to
selected pedal frequencies of 50-55 cycles/min. All subjects will maintain a constant
pedal frequency within this range throughout exercise and the recovery phase. After a 3-
minute warm-up period of unloaded pedaling the workload will be set according to the
individuals functional exercise capacity as derived from the 6MWT. Using standard
encouragement, the exercise will be continued till the point of volitional fatigue or till the
achievement of 80% of the target heart rate (pre-calculated training HR). In the recovery
phase, the patient will continue pedaling to cool down with unloaded pedaling for another
3-minutes.The heart rate will be recorded before and after the warm-up, during
ergometry, at 1 min into recovery period and at end of cool-down period till recovery to
the baseline. A continuous finger plethysmograph tracing will be taken during the
exercise to record the heart rate.
5. Cognitive Parameters
a. Visual Evoked Potential
The Visual evoked potentials will be recorded as per the guidelines of International
Federation of Clinical Neurophysiologists. All the subjects will be briefed and the
procedures were demonstrated before carrying out the actual procedures. Electro-
physiologic studies will be carried out on computerized evoked potential testing
equipment. Ag/Agcl plated disc electrodes will be fixed according to 10-20 international
system on Fz, Pz and Cz sites referred to linked earlobes with a forehead ground, on
predetermined position with the conducting paste. Electrode to skin impedance will be
kept below 5K ohms. The subject will be made to sit 1 m away from the Flash Light and
instructed to fixate on it with the testing eye, while the other eye will be covered with a
patch. Flashes of light will generated by the software; the flashes will be made to at a
frequency of 1 Hz and 300 responses will be recorded and averaged. The absolute
latencies of positive and negative waves will be recorded.
b. Cognitive performance assessment:
Following battery of tests will be performed to assess cognitive performance: Letter
Cancellation Test (LCT), Digit Substitution Test (DST), Visual and Auditory Reaction
Time, Arithmetic Mental Task and Stroop Test

6. Molecular studies
1. Analysis of differential expression of genes related to signal transduction and
metabolic pathways:
Differential expression of gene associated with signal transduction and metabolic
pathway will be studied by Targeted Expression Analysis by Digital RNA
sequencing. RNA will be isolated from blood collected from the patient and
subjected to Digital RNA sequencing. The results of Digital RNA sequencing will
be further validated by Real Time PCR.
2. Assessment of Brain Derived Neurotrophic Factor (BDNF)
Venous blood will be withdrawn at specified time points of the study and stored at
80 C. Serum BDNF will be assessed as a marker for neuronal transmission will
be carried out using specific ELISA kit as per manufacturer guidelines.

EXERCISE TRAINING
Exercise training program will comprise of 90 to 100 minutes of supervised exercise
training for lower and upper limbs, performed over separate sessions three days a week, for
10 weeks. Lower limbs training will include leg-ergometry and treadmill walking. Training
of the upper limbs will include arm-ergometry and free weights. Simultaneous upper and
lower limb training will be performed on Semi-Recumbent Whole Body Exerciser. Exercise
intensity during each session will be incremental and graded according to symptom tolerance
and will be of maximum 20 minutes duration. Patients who desaturate will be trained on
supplemental oxygen at the level evaluated by exercise oximetry.
Analysis of data:
Data will be analyzed using paired T-Test and Repeated Measures ANOVA. The
data obtained at various stages of the study will be compared and a possible correlation shall
be attempted to establish the relationship between the various parameters studied, taking into
account the specific objectives of the study as well as type of the data.
Biodata

1. Name : ANAND PRAKASH YADAV

2. Address for correspondence : House No.-2645, 2nd Floor,


Gali No-7, Bihari Colony,
Shahdara, Delhi 110032

3. Permanent Address : House No. - 532 Kha / 555, Mehndi Tola ,


Behind Police Chowki Dandaiya bazaar,
Aliganj, Lucknow , Uttar Pradesh PIN: - 226022

4. Institution : Department of Physiology,


Vallabhbhai Patel Chest Institute,
University of Delhi
New Delhi-110007
5. Date of Birth : 03-12-1983

6. Gender : Male

7. Category : OBC

8. Whether differently abled : No

9. Academic Qualification

Exam/ Year Board/University/ Subject/Specialization % of Marks

Degree Institute

Ph.D. 2008- 2015 DIPAS (Bharathiyar Life Sciences -


University)

M.Sc. 2007 Allahabad Agriculture Biotechnology 84.44


Institute
B.Sc. 2005 Lucknow University Botany, Zoology, Chemistry 58.8

Intermediat 2001 I.S.C Biology,Physics,Chemistry, 60.6


e
High School 1999 I.C.S.E English. Hindi, Science, 68.8
Maths, Social Science
10. Ph.D thesis title, Guides Name, Institute/Organization/University, Year of Award

Thesis title : Effect of Antarctic Environment on Immune Response of Indian


Expedition Members
Guide Name : Dr. Lilly Ganju
Institute : Defence Institute of Physiology and Allied Sciences
University : Bharathiar University
Year of Award: September 2015.

11. Work experience (in chronological order)

S.No Position held Name of From To Pay Scale


Institute
1 Research Associate Vallabhbhai 1-02-2016 Till Date 49,400
Patel Chest (Consolidate)
Institute
1 Senior Research Defence 26-08-2014 31-01-2016 36,400
Fellow (Post Ph.D Institute of (Consolidate)
Submission) Physiology
and Allied
Sciences

12. Professional Recognition/ Award/ Prize/ Certificate, Fellowship received by the


applicant

S.N Name of the Award Awarding Agency Year


o

1 GATE IIT 2008


2 DRDO Junior Research Fellowship DRDO 2008
3 Appreciation Certificate for the work Ministry of Earth 2010
conducting in Antarctica Sciences
4 1st Prize in Poster Presentation IABMS 2011
Conference
5 Surg Rear Admiral M.S. Malhotra DIPAS, DRDO 2012
Research Prize for Second best
Publication
6 Surg Rear Admiral M.S. Malhotra DIPAS, DRDO 2015
Research Prize for Best Publication
13. Publications (List of papers published in SCI Journals, in year wise descending order).

S.No Author(s) Title Name of Journal Volume Page Year


Immunity against
Himanshi Tanwar,
Pasteurella multocida in
Anand
Animals Vaccinated
PrakashYadav, Journal of Vaccines 10-
1 with Inactivated 2(1) 2016
Brijbhushan, Shweta, and Immunology 14
Pasteurella multocida
Shashi Bala Singh and
and Herbal Adjuvant
Lilly Ganju.
DIPHIP
Anand Prakash
Yadav, Kamla Prasad
Activation of
Mishra , Himanshi Journal of Marine
Complement System
Tanwar, Narendra 5 171-
2 during Ship Voyage and Science Research 2015
Kumar Sharma, Issue-3 179
Winter-over Expedition and Development
Sudipta Chanda,
in Antarctica.
Shashi Bala Singh and
Lilly Ganju.
Evidence for Altered
Metabolic Pathways
Anand Prakash during Environmental
Yadav, Shubhra Stress: 1H-NMR
Chaturvedi, Kamla Spectroscopy based Physiology and 81
3 135 2014
Prasad Mishra, Sunil Metabolomics and behaviour 90
Pal, Lilly Ganju and Clinical Studies on
Shashi Bala Singh. subjects of Sea-voyage
and Antarctic-stay.

Sekar Tamil Selvi Co-administration of


Chitradevi, Gurpreet rIpaB domain of
Kaur, Sivaramakrishna Shigella with rGroEL
Cellular &
Uppalapati, Anand of S. Typhi enhances 12 757-
4 molecular Issue 6 767 2015
prakash the immune responses
immunology
Yadav, Dependra and protective efficacy
pratap Singh, Anju against Shigella
Bansal infection.
Serum HLA-G up-
regulation in stressful
KP Mishra, Anand environment:
Indian Journal of 520-
5 Prakash Yadav and Recombinant HLA-G 140 2014
Lilly Ganju. Protein treated human
Medical research, 523
PBMCs favours pro-
inflammatory cytokines,
Wintering in Antarctica:
Anand Prakash
Impact on Immune
Yadav, KP Mishra, NeuroImmunom- 327
6 response of Indian 19 2012
Lilly Ganju and SB odulation, 333
Expedition Members.
Singh.
KP Mishra, Anand
Antarctic harsh
Prakash Yadav,
environment as natural Indian journal of 357
7 Shweta, Sudipta 27(4): 2012
stress model: Impact Clinical Biochemistry 362
Chanda, Lilly Ganju
on mucosal immunity.
and SB Singh.
Serum levels of
KP Mishra, Anand
immunoglobulins (IgG,
Prakash Yadav,
IgA, IgM) in Antarctic 29
8 Shweta, Sudipta Cellular Immunology 27 2011
summer expeditioners 35
Chanda, D Majumdar
and their relationship
and Lilly Ganju.
with Seasickness.
A Comparitive
KP Mishra, R
analysis of
Mishra, Anand
immunomodulatory Biomedicin & Aging, 2011
Prakash Yadav, B 61-
9 potential of 1
Jayashankar, S Pathology 64
seabuckthorn leaf
Chanda and Lilly
extract in young and
Ganju.
old mice.
KP Mishra, Anand
Ship borne journey
Prakash Yadav,
induces Th1 cytokines Immunological 770-
10 Shweta, S Chanda, L 39 2010
level in Antarctic Investigation 779
Ganju, D Mazumdar
summer expeditioners,
and G Ilavazhagan.
Brijbhushan, Amit Antarctic Environment
Prabhakar, Anand Instigates Activation
Communicated to
11 Prakash Yadav, of Platelets and 2017
Platelets
Mohd. Zahid Ashraf, Inflammatory
Lilly Ganju Cytokines

Technical Expertise:

Heart Rate Variability


Heart Rate Recovery
Functional Capacity Assessment ( Six Minute walk Test and Oximetry)
ELISA
PCR
Handling and taking care of experimental animals
Next Generation sequencing Data Analysis
Maintenance and handling the cultures of cell lines and primary cell
cultures
Toxicological study
SDS-PAGE and immunoblotting
2-D gel electrophoresis
Flow Cytometry
NMR (Metabolomics and Data Analysis)
DNA & RNA isolation
Cloning
Fungal Tip Hyphal Culture
Plant Tissue Culture
Southern Blotting
Biostatistics

Membership of professional Societies:

(1) Life Membership of Indian Immunological Society.

(2) Life Membership of Indian National Academy of Stress Sciences. (INASS)

(3) The Cytometry Society - India.

Other Information:

Participated in 28th Indian Antarctic expedition and stayed for 13 months at Maitri, Indian
Antarctic research base Maitri" and collected largest sample size in a single expedition.
Physiology & Behavior 135 (2014) 8190

Contents lists available at ScienceDirect

Physiology & Behavior


journal homepage: www.elsevier.com/locate/phb

Evidence for altered metabolic pathways during environmental stress:


1
HNMR spectroscopy based metabolomics and clinical studies on
subjects of sea-voyage and Antarctic-stay
Anand Prakash Yadav a, Shubhra Chaturvedi b, Kamla Prasad Mishra a, Sunil Pal b,
Lilly Ganju a,, Shashi Bala Singh a
a
Immunomodulation Laboratory, Defence Institute of Physiology & Allied Sciences, Lucknow Road, Timarpur, Delhi 110054, India
b
Cyclotron & Radiopharmaceutical Sciences Division, Institute of Nuclear Medicine & Allied Sciences, Lucknow Road, Timarpur, Delhi 110054, India

H I G H L I G H T S

Stressful ship journey and long Antarctic stay effects


Tracing of footprints of metabolic changes in difcult environment conditions
1
H NMR metabonomics and biochemical proling in serum
Stimulation of gluconeogenic and energy metabolism related pathways

a r t i c l e i n f o a b s t r a c t

Article history: The Antarctic context is an analogue of space travel, with close similarity in ambience of extreme climate, isola-
Received 16 December 2013 tion, constrained living spaces, disrupted sleep cycles, and environmental stress. The present study examined the
Received in revised form 25 April 2014 impact of the harsh habitat of Antarctica on human physiology and its metabolic pathways, by analyzing human
Accepted 30 May 2014
serum samples, using 1HNMR spectroscopy for identication of metabolites; and quantifying other physiological
Available online 5 June 2014
and clinical parameters for correlation between expression data and metabolite data.
Keywords:
Sera from seven adult males (of median age 36 years) who participated in this study, from the 28th Indian Expe-
Stress response ditionary group to the Antarctica station Maitri, were collected in chronological sequence. These included: i)
1
H NMR baseline control; ii) during ship journey; iii) at Antarctica, in the months of March, May, August and November;
Environmental stress to enable study of temporal evolution of monitored physiological states.
Antarctica 29 metabolites in serum were identied from the 400 MHz 1HNMR spectra. Out of these, 19 metabolites showed
Metabonomics signicant variations in levels, during the ship journey and the stay at Maitri, compared to the base-line levels.
Further biochemical analysis also supported these results, indicating that the ship journey, and the long-term
Antarctic exposure, affected kidney and liver functioning.
Our metabolite data highlights for the rst time the effect of environmental stress on the patho-physiology of the
human system. Multivariate analysis tools were employed for this metabonomics study, using 1HNMR
spectroscopy.
2014 Published by Elsevier Inc.

1. Introduction research in recent years has focussed on the life-style of Antarctic expe-
ditionary members, exposed to harsh environmental and psychological
The Antarctica presents the coldest (temperatures between 25 C conditions over a prolonged period of time [1]. The Antarctic milieu
and 89 C), driest (average RH 0.03%), and windiest (chilly all-year comes with extreme cold, intense UV radiation, white-outs, varying
Katabatic winds averaging 1550 km/h) environment on Earth a magnetic elds, sleep deprivation, altered circadian biorhythms, mem-
continent at the highest average elevation (2300 m above MSL). Due ory loss, and isolation causing signicant physiological and psycho-
to its extreme weather system, and unique geo-location around the logical stresses [24]. They thus prove to be unique human models in
South Pole, it is one of the last frontiers for the human race. Medical a natural laboratory, for studying effects of various stressors on human
health and physiology.
Corresponding author. Tel.: +91 11 23883163; fax: +91 11 23932869. The transient population of the expeditionary teams that winters at
E-mail addresses: lilly.ganju65@gmail.com, lganju@rediffmail.com (L. Ganju). the Indian research base Maitri, comes from densely populated sub-

http://dx.doi.org/10.1016/j.physbeh.2014.05.045
0031-9384/ 2014 Published by Elsevier Inc.
82 A.P. Yadav et al. / Physiology & Behavior 135 (2014) 8190

tropical areas and experiences a sudden exposure to extreme environ- Samples were then collected on-board the ship, after a period of 45
mental stress [5]. During the wintering months they are completely days on-board. Blood samples were subsequently collected during
isolated from civilization, and are deprived of all external life-support their wintering over in Antarctica, in the months of March, May, August
for periods of 8 to 13 months. These attributes singularize Antarctica and November 2009, from the same members, at the Maitri research
as being closest on Earth to conditions in space [2,6]; and provide an station. During the entire duration of the expedition, members were
excellent analogue for long-term space ight [7]. provided with food native to them. None of the subjects used any
A typical biochemical response to acute stress is an increase in the drugs that could signicantly affect the physiological or biochemical
level of plasma corticosterone, glucose, insulin, glycerol, and ketone parameters, or had symptoms indicative of any infection.
bodies (acetoacetate, -hydroxybutyrate, and acetone), as reported by
Ricart-Jane et al. [8]. The response to chronic stress manifests as a 4. Environmental conditions
decreased physical body weight and food intake, enhanced adrenal
weight, and reductions in liver glycogen and plasma insulin [9]. Inter- The geo-location of Antarctica at high latitudes beyond 66S (Tropic
estingly, for both acute and chronic stresses, an overall decrease in of Capricorn), and the low solar altitudes in the region, denes its char-
plasma triglyceride concentrations is common [10]. acteristic frigid and dry climate; and the resultant harsh desert environ-
Earlier studies by VanItallie [9] suggest that physiological response ment, devoid of any vegetation or land based vertebrate life-forms.
to stress is triggered by activation of the sympathetic nervous system Our study started with the 45-day ship journey, during which the
(SNS). The activation of the HypothalamicPituitaryAdrenocortical ship continuously hovered around Larsemann Hills of East Antarctica
(HPA) axis causes release of cortisol from the adrenal cortex, which is (6920 S, 7555 E), where India's new research station was under
responsible for most glucocorticoid activities, marked by increased construction. Further, during the ship-journey, after traversing the
basal metabolic rate, stimulation of glycogenolysis, and mobilization of 40S latitude, the ship underwent prolonged pitching and erratic kinetic
free fatty acids [11]. Our earlier published study [12] shows an increase movements, in the signicantly turbulent waters of the Southern Ocean.
in the level of cortisol during the period of wintering in Antarctica, mea- Three of the seven volunteers suffered severe seasickness, and the rest
sured in the months of March, May and August, in comparison with of the expeditionary team had mild seasickness. Thereafter, during the
the levels of the base-line control. A possible correlation between the stay at Maitri, most of the members suffered with mild nausea and
activation of SNS and HPA axis and the alterations in the metabolite sleep deprivation in the initial phases. Further studies were continued
concentrations during the wintering period is thus suggested by these after de-boarding the ship, during the stay at the Indian Antarctic
studies [13]. Research Station Maitri, at the Schirmacher-Oasis, East Antarctica, at
In the last decade, metabonomics has become a key experimental Latitude 704501.65 S and Longitude 114301.45 E.
method for generating biomedical data. In particular, 1H Nuclear Mag- The Antarctic summer spans from the middle of November to the
netic Resonance (NMR) spectroscopy has the advantage of efciently middle of March, during which period the sun does not set; whereas
obtaining information on a large number of metabolites in serum, the winters begin in the latter half of March, extending until November.
which could serve as a snapshot of the physiological state [14]. 1H Interestingly, from mid-February to April there were variable combina-
NMR spectrum enables simultaneous identication and monitoring of tions of light and darkness in 24-hour cycles, at our location. Complete
a wide range of low molecular-weight metabolites, and provides solar darkness extended from late April to August, while in mid-
the biochemical ngerprint of an organism [1518]. It is now well August to early October there were variable combinations of light and
established that this spectral prole gets altered under stressful darkness again, every 24 h. The duration of this study included: (1)
conditions; and a metabonomic approach can be successfully applied late summer from January to mid-February on-board the ship, in the
to assess the effect of such stressful conditions on the health of an Southern Ocean; (2) entire winter from mid-March to early October
individual [11,19,20]. The present study uses the metabonomics ap- and (3) early summer from October to December, at the Indian Antarc-
proach to interpret 1HNMR spectroscopic data of serum, and follows tic Research base Maitri.
the biochemical variations arising from stress during the ship journey During the summer period, the expedition-members faced constant
to and the chronic Antarctic exposure. Blood pressure and Body 24-hour daylight, and ambient outdoor temperatures ranging from 1
Mass Index of individuals, which served as physiologic markers for C to 10 C. The solar radiation was temperate enough for men,
chronic stress, were also recorded simultaneously, with their blood dressed in moderate clothing, to work with bare hands. However, the
sampling. winters were marked with a phase of absolute darkness, which preclud-
ed any travel or outdoor explorations, and all expedition-members
2. Materials and methods were conned to the interiors of the base. The temperatures reached
minimum levels during the months of May to August, ranging from
The study was approved by the Institutional Ethics Committee (IEC) 25 C to 45 C; making this period the most difcult and stressful
of DIPAS (DRDO, MOD, India); and a written consent was obtained from phase due to environmentally harsh conditions.
each individual included in the study. Necessary permissions from the
National Centre for Ocean and Antarctic Research (NCAOR) and the 5. Sample collection
Ministry of Earth Sciences, Government of India was obtained to
perform the study. The project was sanctioned by DRDO (Ministry of 5.1. Blood collection and serum separation
Defence, India).
The blood was drawn (10 ml) from the voluntary members of the
3. Subjects expeditionary team after overnight fasting, in the morning between 6
and 7 AM and collected in un-heparinized vials, and was incubated for
Seven members (all males; median age: 36 years) of the winter- 1 h at 37 C. The clear serum was collected in small aliquots and stored
ing team of the 28th Indian Scientic Expedition to Antarctica at 70 C, for further analysis.
(200910) volunteered to participate in this study. The team had
undergone pre-departure clinical, psychological, and laboratory ex- 5.2. Conventional biochemical and physiological measurements
aminations to ensure a healthy population for the wintering-over
at the Antarctic. All the biochemical parameters, viz. glucose, cholesterol, triglycer-
For the base-line control, blood was rst drawn from the volunteers, ides, creatinine, SGPT, SGOT and Alkaline Phosphatase, were analyzed
in the month of October 2008, before leaving India for Antarctica. as per manufacturers' instruction (ERBA, MANNHEIM, Germany). Body
A.P. Yadav et al. / Physiology & Behavior 135 (2014) 8190 83

weight and blood pressure were monitored concurrently, whenever the 7. Results
blood samples were collected. BMI was calculated using online tools
[Standard BMI Calculator, National Heart Lung and Blood Institute, US 7.1. Body Mass Index (BMI)
Department of Health and Human Services, USA].
The mean BMI of base-line control was 25.40 2.49, which de-
creased to 23.90 2.41 during the ship journey. After reaching Maitri,
5.3. Sample preparation and NMR spectroscopy the BMI marginally increased to 24.70 2.66 in the month of March;
and then fell to 23.80 1.95 in the month of May, and further to
All the reagents used, including deuterated solvent trimethylsilyl- 22.40 1.03 in August. However, BMI increased again to 25.90
2,2,3,3-tetradeuteropropionic acid (TSP), were from Sigma-Aldrich 1.09 in the month of November (Fig. 1).
(St. Louis, USA). Frozen serum samples were thawed and 500 l of
serum was mixed with 50 l of D2O, and transferred to 5 mm
NMR tubes. External reference was used containing 1 mM TSP in 7.2. Blood pressure (BP)
D2O in closed capillary (Wilmad, USA). All NMR spectra were ac-
quired at the frequency of 400.13 MHz. Bruker-Ultrashield NMR 7.2.1. Systolic
spectrometer at 298 K, and TopSpin 2.1.4 software (Bruker Biospin, The mean systolic BP was 118.00 2.00 mm of Hg for the base-line
Germany) were used for acquisition and processing. After standard controls, which decreased to 115.00 2.88 mm of Hg during the ship
1H 1-D presaturation experiment (zgpr) and cpmg-pr 1-D experi- journey. After reaching Maitri, the Systolic BP increased to 125.00
ment, which selectively reduce the noise due to broad protein 2.70, 124.00 1.76, 126.00 1.60 and 123.00 3.30 mm of Hg; in
signals, acquisitions were recorded and processed as previously re- the months of March, May, August and November respectively (Fig. 2).
ported [21]. The zgpr experiment was used for screening samples,
shimming and identication. CarrPurcellMeiboomGill (CPMG)
spin-echo pulse sequence was used for the suppression of the 7.2.2. Diastolic
water signal with a total spin echo (tau) of 200 ms. Selective irradi- The mean diastolic BP was 80.60 0.66 mm of Hg in the base-line
ation of water resonance was carried out after appropriate relaxation control samples. It decreased during the ship journey to 78.00
delay. For all spectra acquisitions, 64 free induction decays (FID) after 4.10 mm of Hg. After reaching Maitri, the diastolic BP increased to
2 dummy scans were collected into 32 K data points over a spectral 86.00 4.40, 83.00 3.30 and 88.00 1.66 mm of Hg in the months
width of 20 ppm. Acquisition time of 3.98 s was applied. Line broad- of March, May and August. However, in the month of November diastolic
ening factor of 0.3 Hz was used prior to Fourier-transformation, for BP was decreased to 80.00 0.88 mm of Hg (Fig. 2).
processing of spectra. The chemical shifts expressed as delta in the
1
HNMR, were referenced with respect to TSP (delta = 0 ppm).
The peaks identied on the basis of chemical shifts were assigned ac- 7.3. Glucose
cording to previously reported literature [2123], and twenty-nine
serum metabolites were identied based on their characteristic The concentration of glucose at various time points, including before
chemical shifts and multiplicities; and quantied by integrating the Antarctic isolation, is recorded in Table 1. It was observed that during
peak-areas relative to a reference signal from the sodium salt of 3- the ship journey and after reaching to Antarctica the glucose level in-
(trimethylsilyl)-propionic acid (TSP). creased at all time points as compared to the base line level. The base-
line control level of glucose was 97.00 2.70 mg/dl, which increased
to 104.00 3.80 mg/dl during the ship journey. After reaching Maitri
6. Statistical analysis the level decreased in the month of March to 101.30 3.10 mg/dl. How-
ever, during the stay at Maitri, the level increased again to 106.00
For NMR and biochemical parameters values are represented as 3.20 mg/dl in the month of May. This level further increased signicantly
mean standard error (SE). Comparison between results from base- to 108.00 4.70 mg/dl in the month of August with comparison to con-
line, on-board, March, May, August and November months was per- trol (*p b 0.05). However this level decreased in the month of November
formed using the ANOVA. Statistical analysis was conducted using to 100.50 3.50 mg/dl.
SPSS 15 software [IBM Corporation]. A p value of 0.05 was consid-
ered signicant.

6.1. Data reduction and pattern recognition (PR) analysis of 1H NMR


spectra

Principal-Components-Analysis (PCA) is a statistical procedure for


data dimensionality reduction, in multivariate analysis. Statistical signif-
icance analyses were performed using Metaboanalyst-2.0 [www.
metaboanalyst.ca]. From the raw spectra, intensity values in the range
(d 0.210.0 ppm) were tabulated. The region corresponding to suppres-
sion of water signal (d 4.55.0 ppm) was removed during the statistical
analysis as the region is marked with high variability. Data was normal-
ized and mean centered by Pareto scaling. Principal Component (PC)
scores of the concentration of metabolites obtained after integrating
the area under characteristic peaks of different metabolites from the ac-
quired spectra were used for data visualization, where each point on the
Fig. 1. Variation in BMI of Indian Antarctic Expedition-members: before leaving
score plot represents an individual sample at different time points. The India (control), in October 2008; during the ship-journey in December 2008; and
loading plots were also used to detect the metabolites responsible for through the 10-month stay at Maitri Station taken in the months of March, May, August
separation in the data. and November 2009. Data presented as mean SE.
84 A.P. Yadav et al. / Physiology & Behavior 135 (2014) 8190

(p b 0.005 vs. May) in the month of August, it showed an increase


to 196.80 19.50 mg/dl in November (Table 1).

7.6. Creatinine

The concentration of serum creatinine at various time points, in-


cluding before the Antarctic isolation, is recorded at Table 1. It was
observed that during the ship journey and after reaching Antarctica
the creatinine level was higher at all times compared to the base-
line control level of 0.83 0.06 mg/dl. It increased to 1.01
0.079 mg/dl during the ship journey; and further increased signi-
cantly in the month of March to 1.15 0.09 mg/dl (p b 0.005).
Though the level decreased to 0.91 .03 mg/dl in the month of
May, it increased again to 1.04 0.026 mg/dl (p b 0.05) in the
month of August and to 0.99 .029 mg/dl (p b 0.05) in the month
of November.

Fig. 2. Variation of systolic and diastolic blood pressures of Indian Antarctic Expedition- 7.7. SGPT
members: before leaving India (control), during the ship-journey, and through the
10-month stay at Maitri Station taken in the months of March, May, August and The level of SGPT was 31.60 2.50 IU/l in the base-line control
November 2009. Data presented as mean SE. samples, which decreased subsequently at each time point of sampling,
during the entire period of isolation (Table 1).
During the ship journey the level was 27.35 2.60 IU/l. After
7.4. Cholesterol reaching Maitri, the level was 21.30 1.80 IU/l (p b 0.05 vs. control)
in March, and 21.40 1.40 IU/l (p b 0.05 vs. control) in May; which fur-
The analysis of cholesterol in serum of the expeditionary team vol- ther decreased to 19.14 1.60 IU/l in August (p b 0.01 vs. control).
unteers revealed that there was a signicant decrease in the level during However, the level increased to 25.20 1.70 IU/l in the month of
ship journey (149.42 3.10 mg/dl; p b 0.01), as compared to the base- November.
line control level (164.00 3.50 mg/dl). This prolonged decreasing
trend continued even after reaching Maitri in the month of March 7.8. SGOT
(148.70 2.90 mg/dl; p b 0.01). Surprisingly, in the month of May
the level increased signicantly to 203.00 7.90 mg/dl, in comparison Alterations in the levels of SGOT were similar to that for SGPT. The
with base-line control. However, the levels decreased again in the base-line level was 28.40 2.00 which decreased signicantly during
month of August (174.00 2.40 mg/dl, p b 0.01) and November ship journey to 17.40 1.40 IU/l (p b 0.01 vs control). Further the level
(175.00 2.60 mg/dl; p b 0.01). It is noted that signicantly low levels increased to 22.00 1.90 IU/l in the month of March and 22.30
were reached during the ship journey, compared to subsequent levels in 1.06 IU/l in the month of May. The level further decreased to 19.80
May (% p b 0.001), August (^p b 0.005), and November (% p b 0.005) 1.90 IU/l (p b 0.05 vs. control) in the month of August. However, in the
(Table 1). month of November the level increased to 22.40 2.40 IU/l (Table 1).

7.5. Triglycerides 7.9. Alkaline phosphatase

The level of triglycerides was 155.56 6.7 mg/dl in the base-line The levels of alkaline phosphatase remained low throughout the
control samples. The level decreased to 135.80 13.24 mg/dl during expedition, in comparison with base-line control level (Table 1). At
the ship journey; and increased signicantly, after reaching Maitri in base-line, this level was 57.65 4.10 IU/l; and it decreased to 28.85
the month of March, to 221.00 20.16 mg/dl (p b 0.005 vs. control) 3.6 IU/l (p b 0.005 vs. control) during the ship journey, and 31.00
and in May to 232.00 18.35 mg/dl (p b 0.005 vs. control). However, 2.60 IU/l (p b 0.005 vs. control) in the month of March. The levels
while the level decreased signicantly to 162.80 20.30 mg/dl increased in the months of May and August, to 41.70 2.70 IU/l

Table 1
Concentrations of biochemical parameters through environmental stress period of Indian Antarctic expedition members: before leaving India (control), in October 2008; 45 days after
beginning the ship-journey, in December 2008; and through the 10-month stay at Maitri Station taken for the months of March, May, August and November 2009.

Biochemical parameters Transition-period through environmental stress: comparison with base-line (control)

Control Ship March May August November

Glucose 97 2.7 104.3 3.8 101.3 3.1 106 3.2 108 4.7 100.5 3.5
mg/dl
Cholesterol 164 3.5 149.4 3.1 208.7 2.9 173 7.9 170 2.4 175 2.6
mg/dl
Triglycerides 145.6 13.24 135.8 13.3 221 20.16 134 18.35 122 20.3 176.8 19.5
mg/dl
Creatinine 0.83 .06 1.01 .079 1.15 .09 .91 .03 1.04 .026 .99 .029
mg/dl
SGPT 31.6 2.5 27.35 2.6 21.3 1.8 21.4 1.4 19.14 1.6 25.2 1.7
IU/l
SGOT 28.4 2 17.4 1.4 22 1.9 22.3 1.06 19.8 1.9 22.4 2.4
IU/l
Alkaline phosphatase 57.65 4.1 28.85 3.6 31 2.6 41.7 2.7 43.9 2.76 54.3 2.5
IU/l
A.P. Yadav et al. / Physiology & Behavior 135 (2014) 8190 85

(p b 0.01 vs control) and 43.90 2.76 IU/l (p b 0.05 vs. control), respec- 9. PCA analysis of serum samples
tively; but remained signicantly low in comparison with base-line
control. This level further increased to 54.30 2.50 IU/l in November. For systematic representation of metabolic responses to stress,
created by the extreme environmental conditions of Antarctica, a
Principal-Components map was generated from the 1H NMR spectra
8. 1H NMR analysis of serum samples of the serum samples collected from each of the seven individuals at
various time points (Fig. 4a). Most of the variance in the data collected
A number of changes in endogenous metabolites were observed in at all time-points is described by the rst 3 PCs (Fig. 4c). The PCA
1
H NMR spectra of serum collected at various points of time, during score 2D plot of PC1 versus PC2 revealed that each data point,
the expedition. Fig. 3 illustrates representative 400 MHz 1H NMR spec- representing the 1H spectrum of a single individual at different time
tra of serum samples from base-line control, ship-borne journey, and points, and clustered in a way that allowed visualizing a differentiation
after reaching Maitri in the months of March, May, August, and between various stress points in time, in comparison to control. PCA
November. Twenty nine sera metabolites were identied based on of data showed a clear separation between the control sample and
their characteristic chemical-shifts and multiplicities (Table 2); and the samples collected during the ship journey and in the months of
quantied by integrating peak areas relative to a reference signal from March, May, August, and November during the stay at Maitri (Fig. 4a).
3-(trimethylsilyl) propionic acid-d4 sodium salt (TSP). One way From an examination of the PC loading-plot (Fig. 4b), these separations
ANOVA and post-hoc analysis of the quantied metabolites revealed are attributable mainly to the relative increase or decrease in the
signicant elevations (p b 0.05) in the levels of Branched-Chain concentrations of metabolites at different time points. It revealed
Amino-Acids (BCAA), glucose, acetone, creatinine, phosphoric acid, that the main contribution to this separation comes from the in-
D -galactose, aceto-acetate L-tyrosine, and L-leucine. However, signi- creased levels of arginine, BCCA, phosphoric acid, acetone, and D -
cant decreases were observed in lactate, choline and 3-hydroxy buty- galactose, during the ship journey. A further inspection of the load-
rate during the ship journey in comparison with base-line control ing plot revealed that increased concentrations of alanine, glucose,
sample values. After reaching Maitri, in the month of March the levels creatinine, glutamine, creatine and acetone contributed mainly for the
of LDL, alanine and phenyl-alanine increased in comparison with separation, during the months of May and August; while the separation
base-line control and ship journey; while lactate and choline remained during the month of November was mainly because of the increased
signicantly low (p b 0.05). In the month of May, the levels of lactate, level of LDL. However, from the PCA loading plot it was also clear that a
LDL, choline, and BCAA decreased in comparison to the levels at base- decrease in the level of lactate and choline also contributed to the
line control. In the month of August, the levels of alanine, D-galactose, separation from baseline control, at the chosen time points of sampling.
glutamine, and creatine showed signicant increase in comparison
with base-line control, but the levels of LDL, lactate and choline 10. Discussion
remained lower (p b 0.01), in comparison with control. Further,
in the month of November, the level of LDL increased signicantly Utilization of Antarctica's extreme environment as a natural labo-
(p b 0.05). Details of variations in respect of all the identied metabo- ratory has allowed an important expansion in the knowledge of
lites in serum are tabulated (Table 2). human physiology and psychology under extreme stresses [24,25].

Table 2
Concentrations of metabolites measured at various points of transition through environmental-stress of Indian Antarctic expedition members: before leaving India (control), in October
2008; 45 days after beginning the ship-journey, in December 2008; and through the 10-month stay at Maitri Station taken for the months of March, May, August and November 2009.

Metabolites Control Ship March May August November


Nanomolar concentration (nM)

LDL 1722 147 1585 148 1810 251 1458 70 1430 90 2430 171
3 hydroxybutyrate 401 14 505 16 405 31 410 38 407 11 409 22
Lactate 3190 86 2370 102 2430 100 1630 41 1400 50 1110 47
Alanine 1180 51 1380 43 1490 63 1670 113 1890 97 1401 130
Glycoproteins 460 27 378 14 468 26 452 15 455 23 452 31
Aceto-acetate 160 19 100 23 245 27 120 7 220 23 190 16
Succinate 113 12 165 35 140 23 166 43 199 52 139 20
Creatine 206 12 302 18 270 19 310 28 390 36 272 49
Choline 936 26 684 19 620 25 450 25 360 31 270 15
Arginine 930 144 1210 84 1060 77 1050 65 1340 61 1020 57
TMAO 53 4 54 6 53 5 86 16 74 15 79 17
Glycine 300 64 276 62 214 13 113 28 124 19 331 39
Glucose 310 20 510 22 450 24 520 29 780 16 510 34
Phenyl alanine 86 17 116 6 154 17 116 19 94 21 64 20
BCAA 883 31 1746 51 710 68 480 43 574 127 930 56
D-Galactose 310 11 450 50 280 24 380 17 490 32 290 36
Propylene glycol 70 10 120 11 90 13 140 31 150 30 110 18
L-Threonine 150 6 160 27 155 18 140 15 180 35 160 32
Phosphoric acid 320 14 530 62 280 38 330 21 340 44 190 32
L-Lysine 280 16 217 15 274 14 284 57 276 29 277 14
L-Leucine 77 4 49 5 85 10 76 9 81 12 73 6
Acetone 35 5 120 14 40 7 46 7 34 5 40 8
Valine 40 4 30 4 39 11 44 11 50 8 45 6
Citric acid 50 4 64 6 40 4 50 8 44 8 49 10
DSS 25 4 50 10 40 9 45 10 40 4 30 3
L-Histidine 40 7 60 7 45 8 40 5 50 6 35 8
Creatinine 240 18 360 11 390 28 401 14 367 7 334 13
L-Tyrosine 24 5 49 5 40 5 55 7 50 8 40 8
Glutamine 154 13 150 14 159 17 212 10 230 14 147 15
86 A.P. Yadav et al. / Physiology & Behavior 135 (2014) 8190

Control : Base-line (October 2008)

On Ship (December 2008)

At Maitri Station, Antarctica: March 2009


Fig. 3. Representative 1H NMR spectra from the CPMG spin-echo of serum obtained from expedition-members at the base-line control, in October 2008; during the ship-journey, and for
March, May, August and November 2009, through wintering-over at the Maitri Station.

The psychosocial environment of the Antarctic research station pro- studies carried out in Antarctic research stations have indicated
vides a typical research model, which mimics the social element of a that the third quarter of the expedition period (generally indicating
spaceship during a prolonged space ight. Earlier psychological the period between May and August) is the toughest phase that the
A.P. Yadav et al. / Physiology & Behavior 135 (2014) 8190 87

May 2009

August 2009

November 2009

Fig. 3 (continued).

expedition-members encounter [26]. Another study, conducted with base Maitri, situated in the coastal region of East Antarctica, the expedi-
the US Antarctic winter expeditionary group, even indicated that clin- tionary groups face complete solar darkness for a period of four months
ically normal individuals experience signicant depressive symptoms from May to August; bounding the movements of expedition-members
during the peak of the wintering period [26]. At the Indian research to a very limited area, and a constrictive social space.
88 A.P. Yadav et al. / Physiology & Behavior 135 (2014) 8190

The uniqueness of the present study lies in the inclusion of data col-
lected over the 45-day period of sea-voyage in the Southern Ocean and
the 10-month long wintering period at the Maitri Station in Antarctica.
The study is a rst of its kind, tracking the overall impact of the Antarctic
environment on the metabolic and biochemical prole, of seven healthy
humans.
It was observed that BMI, an important physiological index decreased
considerably during the ship journey, with a further signicant decrease
seen until the end of August. This indicated that expedition-members
were stressed during the ship journey, and more specically during the
complete dark phase of the wintering period. Biochemical analysis of
the samples also showed a signicant decrease in the level of triglycerides
(known to decrease during stress) during the ship journey, and then in
the darkness period of May to August; which supports the deductions
from the physiological monitoring of BMI. We have earlier shown [12]
that cortisol increased signicantly until the end of August, further
supporting our present physiological and biochemical ndings. Collec-
tively, we show that subsequent to the initial trigger of rst exposure to
a turbulent ship journey, the wintering over in the months from May to
August was the most stressful period; and that could be the most signif-
icant reason for the changes observed in the metabolic spectra.
SGOT and SGPT are considered as markers for liver-function [27],
while serum creatinine constitutes an excellent indicator for kidney-
function. The decreased SGOT and SGPT levels and the increased
serum creatinine levels indicate that the ship-journey and the long
Antarctic stay negatively inuenced both liver- and kidney-function.
The decreased SGPT and SGOT levels could also be the result of Vitamin
B6 deciency which has been previously reported during the expedition
in Antarctic expeditionary team members [27,28].
A large number of metabolites in body uids can be detected quali-
tatively as well as quantitatively by advanced analytical methods, such
as Nuclear Magnetic Resonance (NMR) spectroscopy, and Mass Spec-
trometry (MS) [17]. Though a majority of metabonomic studies to-
date have focused on proling drug toxicity, an in-depth understanding
of physiological variations consequent to stress inputs is of utmost
importance, and these must be quantitatively estimated to allow for
identication of altered biochemical pathways [2831]. This study
has emphasized on the temporal function of the variability in serum, fol-
lowing exposure to physiological, environmental, and psychological
stresses in Antarctica.
In this study, for the purpose of analysis, the NMR spectral data
derived from serum samples of Antarctic expedition-members was
grouped temporally, according to the time-point of collection, during
the journey to and stay at Antarctica. This facilitated easy identication
of substantial key discriminatory metabolite changes, correlating to
both physiological and psychological stresses induced by the 45-day
long ship journey, and the prolonged period of isolation during the
stay in the constricted environment of the Maitri Station. The PCA and
the Post-hoc ANOVA of the 29 hydrophilic metabolites that were quan-
tied showed that the concentrations of 19 of those metabolites were
signicantly altered, varyingly during different time-periods. This
assisted in data dimensionality reduction, and contributed to the sepa-
ration of samples on temporal scales.
Chemometric analysis of samples collected during the ship journey
indicated increased levels of ketone bodies (3 hydoxybutyrate and

Fig. 4. (a): PCA 2D score plot for data obtained from the concentrations of metabolites
derived from CPMG spin-echo 1HNMR spectra. (0) Red dots represent base line control
October 2008; (1) green dots represent 45 days after beginning ship-journey December
2008; (2) dark blue dots represent March 2009; (3) light blue dots represent May 2009;
(4) magenta dots represent August 2009; (5) yellow dots represent November 2009.
(b): Loading plot for 2D score plot. (c): PCA 3D score plot (0) red triangles represent
the base-line control October 2008; (1) green triangles represent 45 days after beginning
ship-journey December 2008; (2) dark-blue triangles represent March; (3) light-blue tri-
angles represent May; (4) magenta triangles represent August; (5) yellow triangles repre-
sent November 2009. (For interpretation of the references to color in this gure legend,
the reader is referred to the web version of this article.)
A.P. Yadav et al. / Physiology & Behavior 135 (2014) 8190 89

acetone), glucose, arginine, Branched-Chain Amino-Acids, phosphoric in-line with the studies by Bjelland and Tell [35], which showed an
acid, and D-galactose. The increased glucose concentrations could possi- inverse variation between the plasma concentration of choline and
bly be attributed to either hepatic glycogen mobilization, induced by the level of anxiety in the human subjects. Psychological studies on
catecholamines; or to an induced gluconeogenesis via glucocorticoid expedition-members during Antarctic winters show that the levels of
action [32,33]. Another possibility could be a complex combination of anxiety and depression increase greatly during the latter half of the expe-
both the above processes. However, a signicant decrease was observed dition [26]. Choline plays an important role in the synthesis and release of
in lactate and choline levels. Both of these are major raw materials for acetylcholine from neurons [3537]. Free choline is transported by a spe-
gluconeogenesis, and are produced by active skeletal muscle and eryth- cic carrier mechanism across the bloodbrain barrier at a rate that is
rocytes. Therefore, the relative decrease in these metabolites indicates proportional to the concentration of choline in the blood [38]. Studies
stimulation of gluconeogenic pathways following the ship-journey. on age-related memory-loss in adult rats have shown that chronic low in-
This derivation is also supported by the increase in serum glucose levels. take of choline in diet exacerbated this memory loss, whereas choline-
Decreased lactate levels in rats under acute psychological stress have enriched diet resulted in diminished memory loss in adult rats [39,40].
also been reported by Teague et al. [34]. Older mice supplemented with choline-rich diet had changes in the
The observed signicant increase in the levels of serum ketone shape of their neurons in the hippocampus [41]. These ndings suggest
bodies 3 hydroxybutyrate and acetone could be the product of in- that choline also plays an important role in memory function. Thus, the
creased oxidation of long chain fatty acids. It is well known that, signicant decrease in the levels of choline, throughout the expedition
under physiological stress, catecholamines stimulate the hormone sen- duration, could be the causative process for the loss-of-memory suffered
sitive lipases in the white adipose tissue, leading to triglyceride mobili- by the expedition-members.
zation and its oxidation in the liver [8,34]. Thus, this process would be The last phase of the expedition spanned the month of November,
the contributory cause of the increased concentration of serum ketone which is the beginning of Antarctic summers. The average temperature
bodies, and decreased LDL, as a resultant of exposure to signicant at the station increased, and the period was marked by 24-hour day-
physiological stress during the ship journey. light, which facilitated a sense of release from constrained environs,
Thus, in response to tremendous stress of seasickness, the stimula- and a depressive state. The sense of isolation was eased, due to in-
tion of gluconeogenic pathways could result into the observed de- creased long distance visibility. The approaching end of the expedition
creased levels of lactate and alanine, during the 45-day long ship period and the anticipation of a return to familiar environs greatly
journey. reduced psychological stress. This change was clearly reected in de-
The samples collected in March during the stay at Maitri showed creased levels of cortisol, approaching the end of November; shown in
a signicant increase in the levels of alanine and phenylalanine, our earlier published study [12]. PCA analysis and loading plot analysis
compared to the levels at baseline-control and on the ship-journey indicate that a signicant increase in the level of LDL was mainly re-
in inverse correlation to the concentrations in samples collected during sponsible for the separation, based on the qualitatively different emo-
the ship-journey. The levels of lactate and choline also increased in tions during November, which could be correlated with an increased
comparison with the levels during ship journey. This could only be BMI in the expedition-members. Further, the levels of glucose, creatine
explained by assuming a subsequent release of physiological stress at and creatinine were observed to have decreased, in comparison to the
the relatively stable environment of the Maitri Station, initially encoun- levels during the dark months from May to August indicating signi-
tered and accumulated during the ship-journey. cant changes in energy metabolism.
The most interesting phase of the study was through the peak win- Thus, it can be derived from the statistical analysis of NMR data that
tering period months of May to August a period of complete darkness stress caused by extended ship journey and seasickness during the
in the Antarctic continent. This corresponded to extreme environmental transit through turbulent Southern Ocean prompted an alteration in
conditions, low external temperatures, and a phase of isolation from the metabolic pathways, viz. stimulation of gluconeogenic pathways. Fur-
rest of the world. The PCA score plot shows that the May and August ther, during the wintering phase, alterations in energy metabolism, in
time-point variables are closely correlated. A temporal separation of addition to stimulation of gluconeogenic pathways, were observed;
these consists mainly only in increased levels of alanine, glucose, creat- and these could be understood as body's adaptive mechanism to coun-
inine, glutamine, creatine and acetone. A close examination of the load- ter the effects of various factors constituting environmental stress. Fur-
ing plot also yields the observation that the levels of BCAA and LDL were thermore, the BMI can also be considered as a marker of stress, since it
at a minimum during this period, compared to levels at all other points; clearly showed decreased levels during the ship journey and during
while the levels of lactate and choline continued on a signicant down- the winter months of total darkness from May to August indicating
ward slope during this wintering under period of May to August. that this period was the most stressful in the entire extended expedition
An increase in the levels of glucose was also shown by conventional period. The analyses of biochemical tests also clearly suggested that
biochemical tests. This increase in the level could be correlated with the stress affected liver and kidney functions. Thus, a conrmation of
signicant increase in the levels of cortisol in August, as shown in our altered metabolic pathways, caused by being subjected to the stress of
earlier published study [12]. The increase in the level of creatine and Antarctic environment, is concluded.
acetone which are involved in energy metabolism could also be
because of the increased levels of cortisol. Creatinine produced by Acknowledgments
the catabolism of creatine in the muscle and eventually excreted by
the kidneys also showed increased levels; conrmed by biochemical The authors record their sincere thanks to the 28th Indian Antarctic
methods. Decreased levels of BCAA and lactate indicate their uptake Expeditionary Team members who participated voluntarily and
into the TCA cycle in order to increase blood glucose. Thus, the increase allowed their blood to be sampled for this study.
in the levels of glucose, creatine, acetone and creatinine hints that ener- The authors are thankful to the Directors of DIPAS and INMAS,
gy metabolism was stimulated by the HPA axis, during the harsh DRDO, for the support provided for this study.
wintering-over periods. Special thanks to the National Centre for Antarctic and Ocean Research
An increase in the levels of alanine and glutamine, under a long (NCAOR), India for providing all the facilities required for the study.
period of isolation and chronic stress, even where lowered lactate is
observed, is thus likely to be the result of protein catabolism, stimulated
by corticosterone. References
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Original Paper

Neuroimmunomodulation 2012;19:327333 Received: March 5, 2012


Accepted after revision: May 15, 2012
DOI: 10.1159/000339512
Published online: August 1, 2012

Wintering in Antarctica: Impact


on Immune Response of Indian
Expeditioners
Anand Prakash Yadav Kamla Prasad Mishra Lilly Ganju Shashi Bala Singh
Immunomodulation Laboratory, Defence Institute of Physiology and Allied Sciences, Delhi, India

Key Words Introduction


Antarctica Stress Isolation Cytokines IgA
Exposure to different stresses, like extreme cold, heat,
strong magnetic field, UV radiations, high wind velocity,
Abstract altered circadian biorhythms along with psychological
The immune system is one of the major thrust areas in un- stress, has been known to modulate the functionality of
derstanding the effects of adverse climatic conditions on hu- different components of the immune system [13]. The
man health. Exposure to the Antarctic environment, such as healthy, transient population who winter at Indian re-
isolation, cold, UV radiations, magnetic field, blizzards, circa- search base Maitri, Antarctica, generally come from
dian biorhythms, and fear of the unknown, modify various densely populated tropical regions and get suddenly con-
components of the immune system. Members of Antarctic fined to this extreme environment. Most of the expedi-
expeditions suffer significant emotional strain as a result of tioners are totally isolated physically for periods of 813
physical isolation and social deprivation. The present study months that are excellent analogues for long-term space-
was performed on winter team members of the 28th Indian flights [2]. Thus individuals participating in Antarctic
Scientific Expedition. In this study, different immunological expeditions, along with the physiological stress men-
parameters, which mainly include cytokines (TNF- , IFN- , tioned above, suffer significant emotional strain as a re-
TGF-, and IL-4), chemokine MIP-1, immunoglobulins (IgA, sult of physical isolation and social deprivation [4]. Med-
IgM and IgG), cortisol and netrin-1, were assayed in sera by ical research performed for the past 50 years on the dif-
ELISA. Results showed that TNF- and MIP-1 levels were sig- ferent Antarctic Research Expeditions reported certain
nificantly increased in March, May and August while IFN- changes in immune functions [5]. Severe environmental
levels were increased in March and May while TGF- levels stress may have immunosuppressive effects, resulting in
showed a significant decrease in March and May. Serum IgA increased risk for immune-related disorders, such as in-
levels were significantly increased during the entire period fectious diseases, allergy, cancer and autoimmune disor-
of the stressful expedition. Therefore, the present study sug- ders. Muller et al. [6] have reported diminished delayed-
gests that serum IgA could be a potential biomarker for ex- type hypersensitivity to recall antigens in more than 250
treme environmental conditions. study subjects in Antarctica compared with control sub-
45.120.161.219 - 8/22/2016 3:22:42 PM

Copyright 2012 S. Karger AG, Basel

2012 S. Karger AG, Basel Dr. Lilly Ganju


10217401/12/01960327$38.00/0 Immunomodulation Laboratory, Defence Institute of Physiology and Allied Sciences
Fax +41 61 306 12 34 Lucknow Road, Timarpur
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E-Mail karger@karger.ch Accessible online at: Delhi 110054 (India)


www.karger.com www.karger.com/nim Tel. +91 11 23 88 31 63, E-Mail lganju@rediffmail.com
jects stationed on Macquarie Island, which is known for trifuged at 1,200 rpm for 10 min to remove RBCs if any left. The
its harsh winter climate, but where access to the mainland clear serum was collected in small aliquots and stored at 70 C

for further analysis.


is possible. A typical monocytosis was detected and a
striking reduction in the proinflammatory cytokine was Cytokine and Chemokine Assays
noted [7]. Decreased serum levels of IL-10, IL-6, IL-1 re- Cytokines IFN-, TNF- , TGF-, IL-4 and chemokine MIP-
ceptor antagonist [8] and IL-1 were detected [9]. It has 1 assays were carried out using a solid-phase enzyme immuno-
been recognized that cold stress affects various aspects of assay with ELISA kits (Pierce, USA) based on the multiple-anti-
body sandwich principle. The ELISA plates were coated with
both cellular and humoral immunity in experimental an- mouse monoclonal antibody specific for human IL-4, IFN-,
imals: it decreases lymphocyte proliferation, natural kill- TNF- , TGF- and MIP-1 to capture the specific cytokines
er cell count and cytolytic activity; it activates comple- present in the standard and serum. Anti-IL-4, anti-IFN-, anti-
ment and induces heatshock proteins [1012]. Elevation TNF- , anti-TGF- and anti-MIP-1 rabbit polyclonal antibody
of IFN- was reported during exposure to an Antarctic conjugated to biotin was added to each well, followed by strepta-
vidine/HRP incubation for 20 min. At the end, color was devel-
winter [2]. There is also some evidence that psychological oped using peroxide/3,3,5,5-tetramethylbenzidine (TMB) sub-
stress may affect many aspects of the integrative network strate solution. The substrate initiated a peroxidase-catalyzed
between the immune, central nervous and endocrine sys- color change, which was stopped within 15 min by acidification
tems in both animals and humans [13, 14]. The complex with stop solution. Absorbance was measured at 450 nm in an
effects of psychological stress on the interactions among ELISA reader (Biotek, USA).
these three systems need to be investigated in detail. Immunoglobulin Assay
The present study was performed on the winter team Three types of immunoglobulins, i.e. IgA, IgG and IgM, were
of the 28th Indian Scientific Expedition to Antarctica. In measured by the double-antibody sandwich ELISA method as per
this study, different immunological parameters, which the manufacturers instructions (ICL, USA). Briefly, IgA, IgG and
mainly include cytokines, chemokine and immunoglob- IgM were measured in serum by capture ELISA using human im-
munoglobulin reference serum (ICL), goat anti-human IgA, G
ulins, were assayed in serum. Other immunomodulatory and M conjugated to HRP were added. Following washing steps,
signature molecules, like cortisol and the neuronal guide the enzyme bound to the immunosorbent was assayed by addition
molecule netrin-1, known to have anti-inflammatory ac- of a chromogenic substrate, TMB. After 20 min of incubation at
tivity, were also analyzed. Our results show significant room temperature, stop solution (2 N H2SO4) was added. The ab-
alterations in cytokines, immunoglobulins and cortisol sorbance was measured at 450 nm in an ELISA reader (Biotek).
The quantities of immunoglobulins in the test samples were in-
levels in the wintering expeditioners as compared with terpolated from the standard curve constructed from the stan-
their baseline levels. dards and correlated for sample dilution.

Cortisol Assay
Methods Cortisol was assayed in serum using ELISA as per the manu-
facturers instructions (Diagnostic Biochem, Canada). Briefly, an-
Subjects ti-cortisol antibody-coated micro-well plates incubated with 20
Twenty-two members (all males) of the wintering team of the l of either standard or serum samples followed by 100 l of the
28th Indian Scientific Expedition to Antarctica volunteered to cortisol-HRP were incubated on a plate shaker at 200 rpm for 45
participate in this study. Ages ranged from 25 to 60 years, with a min at room temperature. Plates were washed three times with
mean age of 36 years. All had undergone predeparture clinical, wash buffer. At the end, color was developed using peroxide/TMB
psychological and laboratory examinations to ensure a healthy substrate solution which was stopped within 15 min by acidifica-
population for the isolation during the Antarctic stay. The study tion with 1 N HCl. Absorbance was measured at 450 nm in an
was conducted at the Indian Maitri research base, Antarctica. ELISA reader (Biotek).
Blood was drawn from the expeditioners in October 2008 before
leaving India to Antarctica, and in March, May, August and No- Netrin-1 Assay
vember 2009. None of the subjects had any signs or symptoms Netrin-1 was assayed with a kit following the manufacturers
indicative of infection at the time of the study, nor had they used instructions (Cusabio Biotech, China). The microtiter plate pro-
drugs that could significantly affect the immunological parame- vided in the kit was precoated with an antibody specific to ne-
ters. Informed consent was obtained from each of the subjects trin-1. Standards and samples were added to the plate, followed by
involved in the study. a biotin-conjugated antibody preparation specific for netrin-1.
Then a solution of HRP-conjugated avidin was added. The penul-
Serum Separation timate step included addition of TMB substrate. The final step
The blood drawn (10 ml) from the expeditioners was collected included termination of the reaction by addition of 1 N sulfuric
in the morning in unheparinized vials and was incubated for 1 h acid solution. Absorbance was measured at 450 nm in an ELISA
at 37 C. Blood clot formation left a clear serum above, which was
reader (Biotek).
slowly removed and collected in fresh Eppendorf tubes and cen-
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328 Neuroimmunomodulation 2012;19:327333 Yadav/Mishra/Ganju/Singh


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90 a, b, c 120
80
TNF- concentration (pg/ml)

d, e, f

IFN- concentration (pg/ml)


a, b, c
70 100
d, e, f
60 80
50
60
40
30 40
g, h
20 g
20
10 h
0
0 b Control March May August November
a Control March May August November

Fig. 1. Sequential variation in serum TNF- (a), IFN- (b) and 400
TGF- (c) levels in Indian Antarctic expeditioners before leaving
India (control) and during their stay at Maitri, Antarctica, in 350

TGF- concentration (pg/ml)


March, May, August and November. Data are presented as means 300
8 SEs. a ap ! 0.005 March vs. August, bp ! 0.001 March vs. No- a, b f, g, h
vember, cp ! 0.001 control vs. March, dp ! 0.001 control vs. May, 250
ep ! 0.005 May vs. August, fp ! 0.005 May vs. November, gp !
200
0.001 control vs. August, and h p ! 0.05 August vs. November.
d, e
b ap ! 0.005 control vs. March, bp ! 0.001 March vs. August, 150
c
p ! 0.001 March vs. November, d p ! 0.005 control vs. May,
ep ! 0.001 May vs. August, fp ! 0.001 May vs. November, gp ! 100 c
0.05 control vs. August, and h p ! 0.001 control vs. November. 50
c ap ! 0.005 March vs. May, bp ! 0.05 March vs. August, cp !
0.01 control vs. May, dp ! 0.01 control vs. August, ep ! 0.05 May 0
vs. August, fp ! 0.05 control vs. November, gp ! 0.005 May vs. c Control March May August November
November, and hp ! 0.05 August vs. November.

Statistical Analysis the TNF- level significantly decreased to 20 8 3.4 pg/


Values are represented as means 8 SEs. Comparison between ml compared with March (p ! 0.005) and May (p ! 0.005)
results of March, May, August, November and controls were per-
formed using an ANOVA followed by post hoc analysis with Dun- but remained significantly increased compared with the
netts test. The entire analysis was conducted using SPSS 15 soft- control level (p ! 0.001). The level further significantly
ware. p ^ 0.05 was considered significant. decreased to 7.32 8 3.20 pg/ml in November compared
with March (p ! 0.001), May (p ! 0.005) and August (p !
0.05).
Results
IFN- Levels
Serum Cytokines IFN-, a cytokine critical for innate and adaptive im-
TNF- Levels munity against viral and intracellular bacterial infec-
Analysis of serum TNF- in wintering expeditioners tions and for tumor control, was measured in the serum
revealed that the mean baseline control level was 1.51 8 of expeditioners. The baseline control level of serum
1.5 pg/ml (fig. 1a). After reaching Antarctica, the level IFN- was 54 8 5.2 pg/ml (fig.1b). The IFN- level was
significantly increased to 76 8 6.4 pg/ml in March com- significantly increased to 92 8 6.5 pg/ml in March (p !
pared with the baseline level (p ! 0.001). In May, it de- 0.005) compared with the control level. Further, in May,
creased to 66 8 5.5 pg/ml but was significantly increased the level decreased to 83 8 6 pg/ml compared with
compared with the control level (p ! 0.001). In August, March but remained significantly higher than the con-
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Neuroimmunomodulation in Antarctic Neuroimmunomodulation 2012;19:327333 329


Winter Expeditioners
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trol level (p ! 0.005). However, in August, the IFN- lev-
el dramatically decreased to 18 8 2.3 pg/ml, a significant 250

decrease compared with the control level (p ! 0.05), and a, b, c

MIP1- concentration (pg/ml)


the levels in March (p ! 0.001) and May (p ! 0.001). The 200
d, e, f
level further decreased to 3.69 8 2.12 pg/ml in Novem-
ber, a significant decrease compared with the control lev- 150

el (p ! 0.001), and the levels in March (p ! 0.001) and May


100 g, h
(p ! 0.001).
50
IL-4 Levels
The signature cytokine for Th2 immunity is IL-4; it
0
was not detected at any time point of sampling in Indian Control March May August November
Antarctic expeditioners (data not shown).

TGF- Levels Fig. 2. MIP-1 level in Indian Antarctic expeditioners before leav-
The TGF- level was 322 8 32 pg/ml in the baseline ing India (control) and during their stay at Maitri, Antarctica, in
control samples and was significantly decreased at each March, May, August and November. Data are presented as means
8 SEs. ap ! 0.001 control vs. March, bp ! 0.01 March vs. August,
time point of sampling during the entire period of isola- c
p ! 0.005 March vs. November, dp ! 0.001 control vs. May, ep !
tion (fig. 1c). In March, the level was 236 8 16 pg/ml 0.01 May vs. August, fp ! 0.005 May vs. November, gp ! 0.01 con-
(p ! 0.05 vs. control) and was further decreased to 63 8 trol vs. August, hp ! 0.05 August vs. November.
13 pg/ml in May (p ! 0.001 vs. control, p ! 0.005 vs.
March). However, the level was significantly increased to
137 8 21 pg/ml in August compared with May (p ! 0.05)
but it was significantly lower compared with March (p ! reaching Antarctica, the IgA levels were significantly in-
0.05). Furthermore, the level increased to 212 8 33 pg/ml creased at all time points compared with baseline. The
in November compared with August (p ! 0.05) and May baseline control level of IgA, which was 607 8 26 mg/dl,
(p ! 0.05). was significantly increased to 999 8 29 mg/dl (p ! 0.001)
in March. It was further significantly increased to 1,188
MIP-1 Levels 8 65 mg/dl (p ! 0.001 vs. control) in May. However, the
MIP-1, a chemokine generally involved in acute in- IgA level was decreased to 1,005 8 41 mg/dl in August
flammatory states, was measured in the serum samples but remained significantly high compared with the con-
of Antarctic wintering personnel (fig. 2). The baseline trol level (p ! 0.001). It was further significantly decreased
control level was 2.5 8 1 pg/ml. It was observed that after to 859 8 25 mg/dl in November compared with March
reaching Antarctica, the levels initially increased signifi- (p ! 0.05), May (p ! 0.05) and August (p ! 0.05) but was
cantly to 189 8 16 pg/ml in March (p ! 0.001) compared significantly increased compared with the control level
with the control level and then gradually decreased to 162 (p ! 0.001).
8 16 pg/ml in May, but the level remained significantly
higher compared with the control level (p ! 0.001); the IgM Levels
level further significantly decreased to 82 8 10 pg/ml in The concentrations of serum IgM were measured at
August compared with March (p ! 0.01) and May (p ! different time points in Antarctic wintering personnel as
0.01) but remained significantly high compared with the shown in figure 3b. No significant alteration was ob-
control level (p ! 0.01). Further, the levels were drasti- served at any time point before leaving or during the Ant-
cally reduced to 18 8 7 pg/ml in November compared arctic isolation.
with March (p ! 0.005), May (p ! 0.005) and August (p !
0.005). IgG Levels
The concentrations of serum IgG at various time
Serum Immunoglobulins points are shown in figure 3c. The baseline value of IgG
IgA Levels was 1,953 8 54 mg/dl. It increased to 1,995 8 46 and
The concentrations of serum IgA at various time 2,046 8 53 mg/dl in March and May, respectively. The
points are shown in figure 3a. It was observed that after IgG level significantly decreased to 1,830 8 282 mg/dl in
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330 Neuroimmunomodulation 2012;19:327333 Yadav/Mishra/Ganju/Singh


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1,400 400
c, d
1,200 350

IgM concentration (mg/dl)


IgA concentration (mg/dl)

a, b e, f
1,000 300
g, h
250
800
200
600
150
400 100
200 50
0 0
a Control March May August November b Control March May August November

2,500
a

IgG concentration (mg/dl)


2,000

1,500

Fig. 3. Serum IgA (a), IgM (b) and IgG (c) levels in Indian Ant- 1,000
arctic expeditioners before leaving India (control) and during
their stay at Maitri, Antarctica, in March, May, August and No- 500
vember. Data are presented as means 8 SEs. a ap ! 0.001 control
vs. March, bp ! 0.05 March vs. November, cp ! 0.001 control vs. 0
May, dp ! 0.05 May vs. November, ep ! 0.001 control vs. August, c Control March May August November
f
p ! 0.05 August vs. November, gp ! 0.001 control vs. November,
and hp ! 0.05 May vs. November. c ap ! 0.05 May vs. August.

August compared with May (p ! 0.05) and March. The wintering personnel at different time points as shown in
level was further increased to 1,954 8 28 mg/dl in No- figure 4b. No significant alteration was observed at any
vember. time point. The netrin-1 level in the baseline control sam-
ple was 779 8 101 pg/ml and decreased to 614 8 100 pg/
Cortisol Level ml in March. This level increased to 742 8 102 pg/ml in
Cortisol, usually referred as stress hormone, was May and then decreased to 566 8 80 pg/ml in August
measured in the serum samples of Antarctic wintering and increased to 821 8 134 pg/ml in November. The lev-
personnel at different time points (fig.4a). The baseline el of the anti-inflammatory molecule netrin-1 was not
level of cortisol was 14.3 8 1.24 g/dl and increased to significantly altered during the entire isolation period in
18.6 8 2.4 and 19.6 8 2.4 g/dl in March and May, re- Antarctica.
spectively. The level further increased to 24 8 2 g/dl in
August; this increase was significant compared with the
control level (p ! 0.05). However, in November, the level Discussion
significantly decreased to 16.5 8 2.2 g/dl compared
with August (p ! 0.05). In this report, we studied the serum levels of TNF-,
IFN-, TGF-, IL-4, chemokine MIP-1, IgA, IgM, IgG,
Netrin-1 Level stress hormone cortisol, netrin-1 of 22 Indian personnel
Netrin-1, an anti-inflammatory neuronal guide mol- exposed to Antarctic winter. TNF- and MIP-1 levels
ecule, was measured in the serum samples of Antarctic were significantly increased in March, May and August.
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Neuroimmunomodulation in Antarctic Neuroimmunomodulation 2012;19:327333 331


Winter Expeditioners
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30 1,200
a

Netrin-1 concentration (ng/ml)


Cortisol concentration (g/dl)

25 1,000

20 b 800

15 600

10 400

5 200

0 0
a Control March May August November b Control March May August November

Fig. 4. Sequential variation in serum cortisol (a) and netrin-1 (b) levels in Indian Antarctic expeditioners before
leaving India (control) and during their stay at Maitri, Antarctica, in March, May, August and November. Data
are presented as means 8 SEs. a ap ! 0.05 August vs. control, bp ! 0.05 November vs. August.

The IFN- level increased in March and May while, on these immunological studies; however, these findings are
the other hand, TGF- showed a significant decrease in important because there might be long-term health im-
March, May and August; the IgA level increased in Ant- plications. Research is this field is hindered by several
arctic winter in samples compared with their baseline factors, including small population sizes, short lifetime
control levels. exposure to the Antarctic environment and the presence
There are few reports on the cytokine profile of Ant- of multiple variables. Various types of stress have a cer-
arctic expeditioners during short- and long-term stays. tain effect on the Th1/Th2 balance [18]. It is well known
The Australian National Antarctic Research Expedition that an imbalance in Th1/Th2 immunity is associated
(ANARE) has been conducting immunological studies with an increased risk for various immune-related dis-
on personnel staying in Antarctica over the winter (win- eases [19]. However, it has not been established whether
terers) [15]. Work in the 1990s indicated that Antarctic cold exposure induces either a Th1- or a Th2-biased im-
winterers had lowered immunological responsiveness. mune status. Therefore, we were eager to study the effect
One of the studies showed that harsh environmental con- of Antarctic exposure on Th1/Th2 balance, and an anal-
ditions, combined with psychosocial, physical and other ysis of serum IL-4 and IFN- was done. We found that
stresses associated with working and living in physical exposure to an Antarctic winter generated a bias toward
isolation during the Antarctic winter, resulted in altered Th1 immunity.
immune function [16, 17, 25]. Tingate et al. [8] (1997) re- Most of the stress-related studies have shown increased
ported alterations in T cell function, including depres- levels of serum IgA [1921, 24]. Our study also reports
sion of cell-mediated immunity and a 50% reduction in increased sIgA levels in wintering expeditioners com-
T cell proliferation to phytohemagglutinin mitogen in pared with their baseline levels. The possible reason for
addition to altered cytokine production. Shearer et al. [2] increased serum IgA levels is not yet known; however, we
(2002) reported increased proinflammatory cytokines in speculate that generation of IgA is more dependent on T
winter expeditioners, which is in agreement with our helper cells than IgG or IgM [22]. No definite factor is
study. In contrast to our findings, an Italian study showed known so far that might acts as a stimulus for elevation
a marked decrease in serum cytokine levels in human of IgA in Antarctic expeditioners; however, these find-
subjects after prolonged exposure to a cold environment ings do not rule out a possible role of the hypothalamus-
[9]. Shirai et al. [3] have reported that exposure to an Ant- pituitary-adrenal axis response to stress in modulating
arctic winter induced a dramatic decrease in the levels of immunoglobulin production [23].
TNF-, IL-1Ra, IL-6 and IL-1 in serum samples. No We have further investigated the levels of cortisol and
specific patterns of disease have, as yet, been identified by netrin-1 to show the neuro-endocrine immune interac-
45.120.161.219 - 8/22/2016 3:22:42 PM

332 Neuroimmunomodulation 2012;19:327333 Yadav/Mishra/Ganju/Singh


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tion in the serum samples of winter expeditioners and Acknowledgement
found that netrin-1 levels were not significantly altered.
The authors are thankful to all the 28th Indian Antarctic ex-
While cortisol levels were significantly increased during peditioners who have voluntarily given their blood sample for
the winter stay in Antarctica. These data support that ex- conducting this study. Special thanks to the National Centre for
posure to Antarctic winter leads to proinflammatory cy- Antarctic and Ocean Research, India for providing all the facili-
tokine generation. ties required for the study. K.P.M. was summer member and
Hence, our study suggests that overwintering in Ant- A.P.Y. was winter member of the 28th Indian Antarctic Expedi-
tion. The study was supported by DRDO, Ministry of Defence,
arctica affects neuroendocrine immune interaction in fa- Government of India.
vor of Th1 cytokines. Serum sIgA levels could be a poten-
tial biomarker of stress in extreme environmental condi-
tions.

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HM, Smith EO, Lugg DJ: Suppression of hu- man and rat studies. Neuroimmunomodula- 19 Spellberg B, Edwards JE Jr: Type 1/type 2 im-
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Mentors CV

1. Name : DR. VISHAL BANSAL

2. Designation : Assistant Professor

3. Office Address : Department of Physiology,


V. P. Chest Institute,
University of Delhi, Delhi-110007, India

4. E-mail ID : drvishalbansal@hotmail.com

5. Mobile no. : +91-9810525900

6. Educational Qualification: Degrees obtained (From Bachelors Degree)

Degree Institution Field(s) Year

Ph.D. Faculty of Medical Sciences, Physiology 2007


University of Delhi, Delhi

D.N.B. National Board of Examination, Physiology 2000


New Delhi

M.D. Maulana Azad Medical College, Physiology 1998


University of Delhi, New Delhi

M.B.B.S. Maulana Azad Medical College, All Medical Subjects & 1992
University of Delhi, New Delhi Internship

7. Additional Designations:

FCCP : Fellow of American College of Chest


Physicians (ACCP),USA.

MNAMS : Member of National Academy of


Medical Sciences (NAMS), India

8. Discipline / Area of Specialization:

Cardio-Pulmonary Physiology and Rehabilitation


Cardio-Pulmonary Rehabilitation in chronic respiratory patients.
(Clinical services & research)

Measurement of cardio-respiratory parameters and assessment of Airway


and Vascular smooth muscle function in normal and diseased animal
models.
(Experimental research)

1
9. Employment History:

Duration Institution & Duties

Department of Physiology,
V. P. Chest Institute, University of Delhi, Delhi, India

Duties:
Assistant Running Cardio-Pulmonary Rehabilitation program at VPCI.
Professor
(May 2001- Conducting Clinical and Experimental Research
present)
Teaching and training DM, PhD, MD, MSc, BSc & DTCD
students in Respiratory Medicine, Physiology and
Physiotherapy.

Department of Physiology,
Maulana Azad Medical College, New Delhi
Senior Resident
(July 98-May Clinical Research and Teaching
2001) (Medical, dental, nursing, occupational therapy, physical
therapy students)

Department of Physiology,
Maulana Azad Medical College, New Delhi
Junior Resident
(June 95- April Clinical Research and Teaching
98) (Medical, dental, nursing, occupational therapy, physical
therapy students)

Department of Medicine,
L. N. J. P. Hospital, New Delhi

Junior Resident Management and care of In- & Out- PD patients


(Jan 94-Dec94) General and Respiratory Medicine. Clinical Management
(Outpatient, CCU, ICU, Respiratory & Coronary Care Unit,
Emergency Medicine)

10. Research Fellow: Department of Respiratory Medicine


(30th July, 2006 31st July, 2007) West Park Healthcare Centre,
Division of Respirology, Faculty of Medicine,
University of Toronto, Toronto, Canada

2
11. Teaching Experience:

Name of Nature of Post Period Total


the Designation Permanent/ Experience
Institution Temporary From To Yrs. Mths

Maulana
Azad
Junior Residency
Medical
Temporary 5th June, 1995 -
College, 2 10
30th April, 1998
N. Delhi-2

Maulana
Azad
Medical Senior Residency Temporary 1st July, 1998 -
College, 2 10.5
17th May, 2001
N. Delhi-2

V. P. Chest
Institute,
Delhi Assistant Professor Permanent 18th May 2001 15 11.5
University, - Till Date
Delhi

TOTAL TEACHING EXPERIENCE (Till 30th April, 2017) 21 08

12. Guidance of students:


Completed :-
I. DM : 1 (Pulmonary Medicine, Co-supervisor)
II. MD : 2 (Physiology, Supervisor)
6 (Pulmonary Medicine, Co-supervisor)
III. M.Sc. : 3 (Biomedical Science, Co-supervisor)
1 (Physiotherapy {Sports}, Co-supervisor)
Ongoing :-
I. PhD : 1 (Physiotherapy, Co-supervisor)
II. MD : 1 (Physiology, Supervisor)

3
13. External Research Funding:

Funding
Name of the Budget
S. No. Agency and
Investigators Title of the Project (Rs)
Duration
Cardio-Protective Role
Prof. M. Fahim, and Mechanism of Action CSIR
1. 7,62,167/-
Dr. Vishal Bansal of 17 Estradiol in 2003-2006
Anaesthetized Animals
Prof. M. Fahim, Setting up of Patch Clamp
DST (FIST)
2. Prof. K Ravi, Laboratory with Cell 51,70,000/-
2003-2008
Dr. Vishal Bansal Culture Facility
Regulation of Pulmonary
Vascular Tone During DRDO
Prof. M. Fahim,
3. Hypoxia induced 2006- March 14,41,000/-
Dr. Vishal Bansal
Pulmonary 2009
Vasoconstriction
Lipid Reducing Herbal
Compounds CCRUM
Prof. M. Fahim,
4. Provide Protection Nov. 2007-
Dr. Vishal Bansal 13, 31,600/-
Against Diabetes Induced March 2009
Cardiovascular Disorders
ICMR
Prof. M. Fahim Bronchial Reactivity in
5. 2006- March
Dr. Vishal Bansal Diabetic Guinea Pigs 7, 66,938/-
2009
Dr. Dina Brooks The effect of changing
Dr. Kylie Hill, track layout on six-minute Physiotherapy
2,00,000/-
Dr. Roger Goldstein, walk distance (6MWD) in Foundation of
6.
Mr. Tom Dolmage COPD: a Canada
(CAD $
Dr. Vishal Bansal prospective within subject 2007-2008
5,000.00)
study
Dr. Vishal Bansal DIPAS,
Development of exercise
Dr. S. K. Chhabra DRDO
7. protocol to improve
Dr. B. K. Menon Sept., 2014- 50, 00,000/-
hypoxic tolerance
Dr. R. K. Gupta March, 2018

14. Member of Editorial Board & Reviewer for Journals:

(a) Member of the Editorial board of Journal of Krishna Institute of


Medical Sciences (JKIMSU), published by Krishna Institute of Medical
Sciences, Maharashtra, India.

(b) Reviewer for


(i) Current Respiratory Medicine Reviews (CRMR), published by
Bentham Science Publishers, USA.
(ii) Journal for Allergy (J. Allergy), published by Hindawi Publishing
Corporation, USA.
(iii) Indian Journal of Chest Diseases and Allied Sciences (IJCDAS),
published by V. P. Chest Institute, University of Delhi, Delhi-110007.

4
(iv) Human and Experimental Toxicology journal, published by Sage
Publications, USA.
(v) PLOS One, published by Public Library of Open Sciences,
organization, USA.

15. Last five years publications:

i. Jamal Ali Moiz, Vishal Bansal, Majumi M Noohu, Shailendra Nath Gaur,
Mohammad Ejaz Hussain, Shahnawaz Anwer, Ahmad Alghadir. Activities-specific
balance confidence scale for predicting future falls in Indian older adults. Clin Inter
Aging 2017; 12: 645-51.

ii. Vishal Bansal, Shailendra Nath Gaur. Challenges of Developing a Pulmonary


Rehabilitation Programme: Practical Aspects with India as a Model Country. Indian
J Chest Dis Allied Sci. 2016; 58(2): 89-91.

iii. Jamal Ali Moiz, Vishal Bansal, Majumi M Noohu, Shailendra Nath Gaur and
Mohammad Ejaz Hussain. Cross-cultural Adaptation and Psychometric Analysis of
the Hindi-Translated Activities Specific Balance Confidence Scale. Middle East J
Rehabil Health. 2016; 3(1): e34886.

iv. Sunil K. Chhabra, Mansi Gupta, Swapna Ramaswamy, Devi Jyoti Dash, Vishal
Bansal and K. K. Deepak: Cardiac Sympathetic Dominance and Systemic
Inflammation in COPD. COPD 2014; 00 (Online): 1-8.

v. Vishal Bansal & Rajendra Prasad. Pulmonary Rehabilitation in Chronic Respiratory


Diseases. Indian J Chest Dis Allied Sci. 2014; 56:147-8.

vi. Gupta M, Bansal V, Chhabra SK. Abnormal heart rate recovery and chronotropic
incompetence on exercise in chronic obstructive pulmonary disease. Chron Respir
Dis. 2013 Aug; 10 (3):117-26.

vii. Agrawal A, Agrawal A, Bansal V, Pandit M. A systematic approach to interpretation


of heterogeneous lung attenuation on computed tomography of the chest. Lung
India 2013; 30: 327-34.

viii. Vishal Bansal: Pulmonary Rehabilitation: A New Hope For Chronic Obstructive
Pulmonary Disease Patients. Al Ameen J Med Sci. 2011; 4(1): 98-9.