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Indian Journal of Experimental Biology
Vol.52, November 2014, pp. 1098-1105
Modulation of small intestinal homeostasis along with its microflora during
acclimatization at simulated hypobaric hypoxia
Atanu Adak, Kuntal Ghosh & Keshab Chandra Mondal *
Department of Microbiology, Vidyasagar University, Midnapore 721 102, India
Received 4 November 2013; revised 20 May 2014
At high altitude (HA) hypobaric hypoxic environment manifested several pathophysiological consequences of which
gastrointestinal (GI) disorder are very common phenomena. To explore the most possible clue behind this disorder intestinal
flora, the major player of the GI functions, were subjected following simulated hypobaric hypoxic treatment in model
animal. For this, male albino rats were exposed to 55 kPa (~ 4872.9 m) air pressure consecutively for 30 days for 8 h/day
and its small intestinal microflora, their secreted digestive enzymes and stress induced marker protein were investigated of
the luminal epithelia. It was observed that population density of total aerobes significantly decreased, but the quantity of
total anaerobes and Escherichia coli increased significantly after 30 days of hypoxic stress. The population density of strict
anaerobes like Bifidobacterium sp., Bacteroides sp. and Lactobacillus sp. and obligate anaerobes like Clostridium
perfringens and Peptostreptococcus sp. were expanded along with their positive growth direction index (GDI). In relation to
the huge multiplication of anaerobes the amount of gas formation as well as content of IgA and IgG increased in duration
dependent manner. The activity of some luminal enzymes from microbial origin like α-amylase, gluco-amylase, proteinase,
alkaline phosphatase and β-glucuronidase were also elevated in hypoxic condition. Besides, hypoxia induced in formation of
malondialdehyde along with significant attenuation of catalase, glutathione peroxidase, superoxide dismutase activity and
lowered GSH/GSSG pool in the intestinal epithelia. Histological study revealed disruption of intestinal epithelial barrier
with higher infiltration of lymphocytes in lamina propia and atrophic structure. It can be concluded that hypoxia at HA
modified GI microbial imprint and subsequently causes epithelial barrier dysfunction which may relate to the small
intestinal dysfunction at HA.
Keywords: Epithelial barrier, Growth direction index, Hypoxia, Microflora, Small intestine, Systemic inflammation
Mountains cover one-fifth of the earth’s surface; 38
million people live permanently at altitudes ≥2400 m,
and 100 million people travel to high-altitude
locations each year1. During ascent from sea level to
high altitude (HA), barometric pressure of atmosphere
falls exponentially that decreases the partial pressure
of oxygen in the inspired gas of human. People like
military personnel, veterans, athletes and travellers
generally face such environmental hazards above
1,493 m and extreme altitudes above 5,486–6,096 m
during acclimatization2,3
. It causes physiological
hypoxia in which an individual face an initial
“struggle response” that induced several neurological
complications collectively known as acute mountain
sickness (AMS)1,2
. Moreover, an individual exposed
to other environment stressors like ultraviolet
radiation, lower temperature and inability to maintain
adequate personal hygiene and isolation from
adequate medical care complicate the AMS. Apart
from neurological and pulmonary syndrome many
sojourners also experience several gastrointestinal
(GI) disturbances like anorexia, epigastric discomfort,
flatus expulsion, dyspepsia, nausea, severe acidity,
vomiting, infectious diarrhoea and haematemesis
etc4,5
. The wide mucosal surface (200-300 m2) of GI
tract harbour and establish complex microbial
ecosystem combining the gastrointestinal epithelium,
immune cells and its resident microbiota. The
microbiota is the most important and integral part of
GI tract that participate in digestive, protective,
structural and metabolic homeastasis5. A number of
digestive enzymes secreted by microbial imprint
hydrolyse all undigested or semi-digested food
into absorbable form. The disturbance of GI
microenvironment and its ecosystem is related to the
weakening physio-chemical barrier through the
induction tone of the local inflammatory responses4,6
.
——————
*Correspondent author
Telephone: 03222-276554/555 (ext. 477)
Fax- (91) 03222-275329
E-mail: mondalkc@gmail.com
ADAK et al.: SMALL INTESTINAL HOMEOSTASIS & HYPOBARIC HYPOXIA
1099
Still the relationship between intestinal dysfunction
with microbial modification during hypobaric hypoxia
is not elucidated. The present study has been
undertaken to investigate the detrimental effects of
hypobaric hypoxia at HA on GI microflora, in male
albino rats model which were exposed to a simulated
hypobaric hypoxic condition. Modulations of some
indicator predominant microbial populations have
been examined to assess pathophysiological status of
small intestine.
Materials and Methods Experimental animals and grouping—Healthy
male albino rats (30; average body weight 145 ± 7 g)
were used. The rats were divided randomly into
following two groups of 15 each: hypobaric hypoxic
(HH) and normabaric (NB) group. All animals were
housed in polypropylene cages (34 × 28 × 19 cm3)
with normal 12: 12 h L:D cycle. The animals were
acclimatized for a week in laboratory environment.
They were not under treatment of any medication
and allowed to consume specific boiled homogenous
diet (containing carbohydrates, 74.05%; proteins,
10.38%; fibre, 2.20%; iron, 56 ppm; calcium,
400 ppm and sodium, 500 ppm) throughout the
experimental period and water ad libitum4.
Exposure to hypobaric hypoxia and sample
collection—The rats of hypobaric hypoxic (HH)
group were exposed to 55 kpa (~4872.9 m altitude)
for 8 h/day consecutively for 30 days at the same day
time in a simulated hypobaric hypoxic chamber with
adequate supplement of food and water. The NB
group was maintained at normal atmospheric pressure
(101.3 kPa) without interrupting their normal activity
and circadian rhythm. At 10 days interval 5 rats in
each group were sacrificed by deep anaesthesia
with chloroform and part (2-3 cm) of the ileum
was dissected aseptically4. All procedures were
performed in accordance with the animal care
guidelines of Vidyasagar University, 3rd
meeting held
on 25.09.12 (item no. 3.ii).
Enumeration of microbial population—The
contents of ileum segments were suspended in
sterilized phosphate-buffer saline (PBS; pH 7.0 and 9
g/L NaCl) and it was vortex thoroughly. The
suspension was used and some indicator cultivable
bacterial populations in the ileum were enumerated by
standard spread plate technique in the selective media
following the instructions of the HiMedia manual
(www.himedialabs.com). The total aerobes and
anaerobes were cultured on single-strength trypticase
soya agar and reduced wilkins chalgren agar
(supplemented with sodium succinate, hemin,
vancomycin, menadione, oleandomycin phosphate
polymyxin B and nalidixic acid). The population
density of Escherichia coli, Bacteroides sp. and
Clostridium perfriengens were enumerated by
culturing on Mac-Conkey, bacteroides bile esculin
agar (supplemented with gentamicin 100 mg/L) and
perfringens agar base (supplemented with supplement
I = sodium sulphadiazine 100 mg/L and supplement
II = oleandomycin phosphate 0.5 mg/L and
polymyxin B 10 000.0 IU/L). Bifidobacterium
sp., Lactobacillus sp. and Peptostreptococcus
sp. were cultivated respectively on bifidobacterium,
rogosa SL and KF streptococcal agar. Anaerobic
conditions were maintained in a CO2 incubator [Heal
force Air jacket (HF 151 UV, 1 unit)], filled with 10%
CO2 and H2 and the population density was expressed
as logarithm (10 base) value of colony forming unit
(CFU)/g of wet ileum content4,5,7
.
Growth direction index (GDI)—The CFU/g of
bacterial populations in logarithmic values of NB
group tallied with the HH group. When control
log10CFU/g was higher in comparison to test
log10CFU/g, the GDI was denoted as negative and
reverse event was followed as positive. This will
show a straight forward portrait about expansion and
contraction of a microbial population in a particular
ecosystem7.
Measurement of gas volume—The ileum content
(100 µL) was added in 30 mL of Mac-Conkey broth
and volume of the gas was measured by an inverted
volumetric Durham’s tube after 24 h of incubation at
37 °C. Gas formation ability was expressed in mL/g
of ileum content5.
Faecal enzymes activity—The faecal aliquot was
centrifuged at 10,000 rpm for 5 min, the supernatant
was collected and α-amylase and gluco-amylase
activity was determined by glucose oxidase-
peroxidase and dinitro salicylic acid method8.
Proteinase, alkaline phosphatase and β-glucuronidase
activity were assayed with some modifications9,10,11
.
Proteinase activity was assayed by using 0.8 mL of
1% (w/v) azocasein as the substrate and 0.2 mL of
faecal aliquot and incubated for 45 min. The reactions
were stopped by equal volume of 5% (w/v)
trichloroacetic acid and 1 mL of 1 (N) NaOH was
added, the absorbance was taken at 450 nm. Alkaline
phosphatase activity was assayed by using 0.1 mL of
50 mM para-nitrophenylphosphate and 50 µL faecal
INDIAN J EXP BIOL, NOVEMBER 2014
1100
aliquot and 0.7 mL of 50 mM tris buffer (pH 8.5) and
incubated for 1 h at 37 °C. The reaction was stopped
by 0.1 mL of 1(M) perchloric acid and 2.5 mL of 0.50
mM NaOH. The absorbance was taken at 420 nm.
The activity of β-glucuronidase was determined by
using 0.7 mL of 1.2 mM phenolphthalein glucuronide
and 0.7 mL of 100 mM sodium acetate buffer
(pH 3.8) and 0.1 mL faecal aliquot. It was mixed and
incubated at 37 °C for 30 min. Thereafter 5 mL of
200 mM glycine buffer (pH 10.4) was added and OD
was taken at 540 nm. Total protein in the faeces was
estimated and the enzyme activities were expressed in
specific activity (U/mg of protein)12
.
Analysis of antioxidant parameters—The dissected
segment of ileum was transferred in a chilled
phosphate-buffer saline (PBS; pH 7.0). The inner
content was gently washed thereafter it was opened
vertically and epithelial cells were scraped out with a
scalpel. The scraped cell (~10 mg) was suspended in
chilled PBS (pH 7.0) and cell membrane was
disrupted by sonicator [SONAPROS-PR 250 MP].
The sonication was performed with a titanium horn
(3 mmφ) at 20 kHz for 15 min at pulse rate of 2 sec
at 15% amplitude. The temperature was maintained
at 30 °C throughout the operation process by
surrounding the suspension in ice bath. The
suspension was centrifuged at 10,000 g and the
supernatant was collected. Activities of antioxidant
parameters like superoxide dismutase (SOD), catalase
(CAT) were measured by studying the inhibition of
pyrogallol auto-oxidation and catalysis of H2O2 at 420
and 240 nm13,14
. The reduced glutathione (GSH) and
oxidized glutathione (GSSG) were determined using
enzymatic recycling method15,16
. Lipid peroxidation
level was evaluated by the thiobarbituric acid reaction
for malondialdehyde (MDA) formation at 532 nm17
.
Histological study by light microscopy—About 2-3
cm of isolated ileum part was fixed in the Carnoy’s
solution (60% ethanol, 30% chloroform and 10%
glacial acetic acid). After 2 h fixation, tissues were
dehydrated in graded alcohol and embedded in
paraffin and cut into 3-5 µm thin sections4,6
. After
periodic acid Schiff (PAS) staining, intestinal
morphology like villus height, goblet cells number,
tissue debris in lumen and modification of mucosal
layer was observed using a Nikon Eclipse LV100POL
polarized light microscope.
Scanning electron microscopic analysis—Fresh
small intestinal tissue was rinsed with cold saline
(0.9% NaCl) and cut into 5 mm × 5 mm sections,
fixed in 2.5% glutaraldehyde, 10% osmium and
dehydrated in sucrose solution containing PBS. Then
it was gold coated and observed under scanning
electron microscope (FEI Quanta -200 MK2). The
arrangement of microvilli, deformed and exfoliated
villi and the intercellular space between epithelia were
examined4,6
.
Immunoglobulins assay—The luminal content (20
µL and 10 µL) was mixed with the 500 µL of
activation buffer and 50 µL anti- rat IgA and IgG
antibody was added respectively4. After 5 min of
agglutination reaction turbidity was measured at 340
nm and the concentration of IgA and IgG was
expressed as mg/g of the intestinal content.
Statistical analysis—The data were presented as
the arithmetic mean of five replicas (mean ± SD).
Variations in microbial count were examined by one-
way ANOVA (Kruskal-Wallis)7. Significant variation
was measured by using Sigmaplot 11.0 (USA)
software and accepted at the level of 5 and 1%
(P<0.05 and P<0.01).
Results
During experimental period no morbidity of rat
was recorded. The total aerobes in the ileum content
were 4.75 that decreased to 2.61 with negative GDI of
1.81 after 30 days of acclimatization at 55 kPa. This
reduction was 138 folds in respect to the population
size of NB group (Table 1). In contrast, the quantity
of total anaerobes significantly (P<0.05) increased
from 6.02 to 8.04 (log10CFU/g) with positive GDI
(1.33), which seemed to be 105 folds higher than the
control population size. After 30 days of hypobaric
hypoxic stress the population ratio of total aerobes
and anaerobes changed from 1:1.26 (ratio of
log10CFU of the population) to 1:3.08 in HH group.
The facultative anaerobes, E. coli group conquered
with population density of 2.76 in NB group
significantly enlarged 26 fold with a positive GDI of
1.51 in HH group after 30 days hypobaric hypoxic
stress. The population density ratio of E. coli, total
anaerobes and total aerobes was 1:2.18:1.72 (ratio of
log10CFU/g of NB group) which altered to 1.6:3.08:1
(ratio of log10CFU/g of HH group) at 30th day of
hypoxic stress. In NB group the strict anaerobes like
Bifidobacterium sp., Bacteroides sp., and
Lactobacillus sp. were present with population
density of 2.32, 3.12 and 4.93. After HH treatment
these were modified to 2.19, 4.59 and 5.21
respectively in HH group. Among these groups
ADAK et al.: SMALL INTESTINAL HOMEOSTASIS & HYPOBARIC HYPOXIA
1101
Bacteroides sp. and Lactobacillus sp. inflamed 30 and
2 fold with an uprising GDI of +1.47, +1.05 whereas
Bifidobacterium sp. increased 1 fold with a GDI of
+1.05 in HH group after 30 days (Table 1). Therefore
population ratio of Bifidobacterium sp., Bacteroides
sp., and Lactobacillus sp. was 1:1.42:2.25 (ratio of
log10CFU/g of NB group) that adjusted to 1:1.97:2.24
(ratio of log10CFU/g of HH group) on 30th day of
hypoxic stress. Obligate anaerobes like C. perfringens
and Peptostreptococcus sp. was 4.30 and 5.36 in NB
group which expanded to 6.01 and 6.85 in HH group
with a positive GDI of 1.39 and 1.27. Finally, the
population density ratio of C. perfringens and
Peptostreptococcus sp. was 1:1.24 (ratio of log10
CFU/g in NB group) which changed to 1:1.13
(ratio of log10CFU/g in HH group) due to enlargement
of population size by 51 and 31 fold during 30 days
acclimatization at hypobaric stress.
The capability of gas-formation by composite
microflora in NB group was 6.5 mL/g that
polynomially (R2 = 0.97) increased to 22.6 mL/g after
30 days of adaptation at hypoxia.
Apart from higher gas formation in ileum,
basal activity of some microbes associated enzymes
like α-amylase, gluco-amylase, proteinase and
β-glucuronidase enhanced in HH group after 30th day
of familiarization to the simulated hypoxia (Table 2).
The levels of IgA and IgG in the luminal content
were 1.9 and 3.06 mg/g (in NB group) that
progressively increased to 6.89 and 8.27mg/g after
30 days of hypobaric hypoxic adaptation.
The activity of SOD was 106.35 U/mg of protein
(in NB group) of small intestinal epithelia that
significantly (P<0.05) decreased to 32.26 U/mg of
protein during adaptation 30 days in hypoxia (Table 3).
The CAT activity was 238.27 U/mg of protein in NB
group. It also significantly (P<0.05) reduced
respectively to 165.54, 103.62and 79.83 U/mg of
protein after10th 20
th and 30
th day in HH group.
Consequently, the activities of SOD and CAT
Table 1—Alteration of some indicator bacterial populations of small intestine and its change with GDI after
30 days of hypobaric hypoxia
[values are mean ± SD]
Hypobaric hypoxic exposure duration (in days)
NB group HH group
Microbial parameters
10 20 30
GDI
(log10NB/log10HH30) and folds
Total aerobes 4.75±0.14a 3.54±0.11b 2.56±0.09c 2.61± 0.06c -1.81,138
Total anaerobes 6.02±0.17c 6.19±0.15c 7.41±0.13b 8.04±0.11a +1.33,105
Escherichia coli 2.76±0.03d 3.55±0.08c 4.01±0.06b 4.18±0.08a +1.51,26
Bifidobacterium sp. 2.19±0.04c 2.15±0.06c 2.17±0.04bc 2.32±0.07a +1.05,1
Bacteroidetes sp. 3.12±0.09d 3.98±0.11c 4.32±0.08b 4.59±0.09a +1.47,30
Lactobacillus sp. 4.93±0.07c 4.95±0.10c 5.01±0.11bc 5.21±0.13a +1.05,2
Clostridium perfriengens 4.30±0.09d 5.22±0.08c 5.75±0.11b 6.01±0.13a +1.39,51
Peptostreptococcus sp. 5.36±0.12d 5.63±0.11c 6.31±0.14b 6.85±0.15a +1.27,31
The population density of different bacteria was expressed (mean of 5 replicates in log10CFU/g±SD). Superscripts (a, b, c, d) in a row are
significantly different at P<0.05.
Table 2—Modification of some luminal enzymes activities in NB group and HH group during 30 days hypobaric hypoxic
acclimatization
[values are mean ± SD]
Hypobaric hypoxic exposure duration (in days)
HH group
Enzyme activity
NB
10 20 30
Increase after
30 days over
NB
(%)
α-amylase 305.22±12.11c 313.12±13.45c 387.29±15.11b 469.34±16.57a 53.77
Gluco-amylase 127.66±6.12d 143.32±6.75c 188.16±7.33b 205.47±9.15a 60.95
Proteinase 7.76±0.87b 5.26±0.35c 8.19±0.91b 12.84±1.22a 65.46
β-glucuronidase 3.42±0.34b 3.95±0.38bc 4.14±0.44b 5.85±0.48a 71.05
Alkaline phosphatase 6.39±0.25d 7.43±0.34c 8.56±0.51b 9.77±0.87a 52.89
Specific activities of enzymes were expressed as U/mg of protein. Superscripts (a, b, c, d) in a row are significantly different at P<0.05.
INDIAN J EXP BIOL, NOVEMBER 2014
1102
activity, GSH and GSSG pool in the small intestinal
epithelia were declined gradually after 10th, 20
th and
30th day in HH group. In relation to lower level of
antioxidants the formation of MDA in small intestinal
epithelia in HH group was progressively elevated
(P<0.05) from 3.17 mM/mg of protein (in NB group)
to 8.82 mM/mg of protein in HH group after 30 days
acclimatization in hypoxia.
Histological study of small intestine by PAS
staining showed that thinner layer of mucus along the
surface of villi with reduction of goblet cell in HH
group (Fig. 1). Besides, the villus length of small
intestine shortened accompanying disordered
arrangement and edema with higher infiltration of
inflammatory cells in lamina propia. Tissue debris in
the lumen of small intestine was noted in the HH
group but there were no such noxious and debris
matter present in NB group. Scanning electron
microscopy also revealed severely injured intestinal
mucosa as well as evident atrophy and disordered villi
in HH group in respect to orderly intestinal villi of
NB group (Fig. 2).
Discussion Acute exposure to the hypoxic atmosphere at high
altitude (above 8,000 metres) stimulated the
sympatho-adrenal and central nervous system that
cause in pathophysiological and metabolic
malfunctions1,18
. The role of enteric microbiota in
bidirectional gut-brain interaction in health and
disease has been reported. The intricate network of
commensal bacterial flora and GI tract is co-evolved
toward a mutually beneficial state19
.
Normally, the proximal part of small intestinal
mucosa was colonized with 107-10
9 number different
(300-500) microbial species that formed a stable
alliance with the host by a predominance of gram-
Table 3—The activity of SOD, catalase, GSH, GSSH and MDA level of the ileum epithelia of the NB (101.3kPa) and HH (55kPa) group
at different days (10, 20 and 30th day) of hypobaric hypoxic stress
[values are mean ± SD]
Hypobaric hypoxic exposure duration (in days)
HH group
Antioxidant parameters
NB group
10 20 30
SOD (U/mg of protein) 106.35±7.11a 76.55 ±3.25b 51.43±2.33c 32.26 ±1.74d
Catalase (U/mg of protein) 238.27±10.21a 165.54±10.66b 103.62±4.91c 79.83±3.26d
GSH (µmol/mg of protein 18.45±0.91a 16.29±0.88b 12.38±0.76b 11.79±0.54c
GSSG (µmol/mg of protein ) 0.10±0.001d 0.17±0.003c 0.25±0.004b 0.27±0.004a
GSH/GSSG 185.0±9.700a 95.82±4.21b 49.52±2.13c 43.66±1.78c
MDA (mM/mg of protein) 3.17 ± 0.11d 6.78±0.26c 8.15±0.28b 8.82±0.39a
Values within a row followed by superscripts (a, b, c, d) significantly different at P<0.05.
Fig. 1—Histological examination of rat small intestine under light
microscope by PAS staining [The mucosal layer with intact epithelia,
arranged villi, higher crypt depth and goblet cells with less
lymphocyte in lamina propria in NB group (A), disordered villi, lower
crypt depth with higher infiltration of lymphocytes in lamina propria
accompanying irregular morphology and disorganised, necrotized
epithelia in HH group (B). Arrow indicates the significant changes]
ADAK et al.: SMALL INTESTINAL HOMEOSTASIS & HYPOBARIC HYPOXIA
1103
negative organisms and anaerobes19
. The
environmental stress at HA caused hypoxia, that in
turn reduced the oxygen delivery to peripheral tissue
and cause cellular dysoxia2,5
. This higher anaerobic
state of epithelia decreased the population of total
aerobes. But it favoured the growth of total anaerobes
in luminal microenvironment. The metabolic and
respiratory networks of prokaryotes are drastically
modulated by ambient oxygen tensions however
micromolar concentrations of oxygen are sensed
and transduced into changes in gene expression20
.
In E. coli there is a graded series of responses which
facilitated its growth from microaerobic to aerobic
range20
. This remarkable adaptability and aerotaxis
ability of E. coli induced to increase in population
size at the altered hypoxic microenvironment.
This proliferation regulated the growth of other
anaerobes like Bacteroides sp., Lactobacillus sp and
Bifidobacterium sp. But it could not be confirmed
from the experiment that why the enlargement of
Bifidobacterium sp. was lower than other microbial
population. Higher colonization of obligate anaerobes
in the lower oxidation reduction potential of small
intestine encouraged the establishment of other strict
anaerobes like C. perfriengens, Peptostreptococcus
sp. This modified microbial imprint in the small
intestine may influence the adhesive and invasive
capacity with epithelial cell and caused opportunistic
infection during acclimatization in hypoxemia.
Asides, hypoxia causes an immediate increase in
gastric vagal nerve activity exerted a tonic inhibitory
effect which in turn decrease the gastric tone and
amplitude of contraction21
. The lower gastrointestinal
motility and reduced delivery of gastric content
resulted in decrease of absorption and self cleaning
ability that extended the stay time in a nutrient rich
environment21
. It may encourage proliferation of
different opportunistic pathogenic bacteria as well as
immuno modulation of epithelia. In a rat model, the
migrating motor complex, often referred to as the
housekeeper of the gut, was critical to the prevention
of bacterial overgrowth in the upper small bowel22,23
.
Depletion of oxygen helped over population of different microbes that involved in higher anaerobic digestion and produced excess flatus and acid in the small intestine
4.
The microbial population secrete an array of enzymes to digest the unabsorbed dietary substance to salvage energy and provide it to the epithelia. Malabsorption of nutrients in hypoxia induced the activity of α-amylase, gluco-amylase by which overpopulation of microbes struggled to harvest energy and survive in the diverged ecological niche
5.
Besides, protein was also utilized as a nitrogen source from which less energy and more toxic metabolites like urea and ammonia were produced. It may diffuse through the epithelial barrier and enter into the systemic circulation and complicated the acute mountain sickness (AMS). Moreover higher activity
Fig. 2—Morphological study of the surface of small intestine by
scanning electron micrograph [Arranged projection of villi with
intact in NB group (A), severely injured, smooth villus projection
and distorted mucosa along with necrotized tissue and disordered
villi with exfoliated microvilli in HH group (B). Arrow indicates
the significant changes]
INDIAN J EXP BIOL, NOVEMBER 2014
1104
of bacterial β-glucuronidase may cleave acyl glucuronides to their aglycone that reabsorbed through the hepatobiliary system and increase the load of carcinogen in the lumen of small intestine
5.Therefore, consumption of any nonsteroidal
drugs for the management of hypobaric hypoxia may initiate a cascade of events leading to epithelial damage and inflammatory responses which is triggered by higher permeability of the gut mucosa. These were also observed in the human gastrointestinal tract during high altitude acclimatization for 15 days at Leh (~3,505 m)
5. But it
was not clear from the experiment whether the sole source of these enzymes was either microbial or rat intestinal epithelia. Consequently, burden of lipopolysaccharide (LPS) due to enlargement of gram negative bacterial population heightened the level of alkaline phosphatase that removes the phosphate from glutamine of the lipid moiety to neutralize this LPS toxicity
24. The activation of local innate immune
activity stimulated the local gut-associated immune system which secretes immunoglobulin like IgG and IgA in the gut lumen that was also observed in human and rat model during acclimatization in hypoxia
2,4,5.
Further, hypoxemia induced microvascular
dysfunction during HA acclimatization altered
cellular metabolic pathways which likely results in
ATP depletion, acidosis and altered ion pump activity.
It reduced cellular viability and increased paracellular
permeability and produced excessive reactive oxygen
species (ROS)4,6,25
. This overwhelmed of ROS
declined the SOD and CAT level of the intestinal
epithelia (Table 3) with lower redox pool of
GSH/GSSH. Therefore, unpaired electrons of the
ROS damaged the lipid bilayer membrane of
intestinal epithelia and resulted in higher formation of
MDA in the small intestinal epithelia.
The discontinuous layers of mucus on small
intestinal epithelia confer various ecological functions
to the indigenous GI microflora. Normally, it
lubricates the lumen and acts as innate immuno
barrier to protect against the mechanical, chemical
injury and bacterial invasion22
. The composition and
expression was changed in broad range of luminal
insults, including changes in the normal microbiota.
The reduction in number of goblet cells in HH group
indicated that hypobaric hypoxia caused cytoclasis of
goblet cells and dysfunction of the immuno barrier.
Besides, higher infiltration of inflammatory cells in
lamina propia, shortening of villus length and tissue
debris in lumen of HH group indicated the induction
of severe inflammation, damage and necrosis of the
mucosal layer of intestine. This injury conferred the
opportunity to pathogenic microbiota to adhere and
invade in the systemic circulation and may complicate
the AMS during hypoxic acclimatization. The SEM
also revealed intestinal mucosal atrophy, mucosal
barrier dysfunction that also strongly correlated with
oxidative damage of epithelial layer.
Conclusion
It can be concluded from the results that hypoxic
stress modulated indigenous GI microflora and the
magnitude of association with host. The expansions of
anaerobic bacterial populations coupled with the
higher acid and gas formation in ileum. Distortions of
microbial ecology and its functional activities in
ecological niche, change luminal microenvironment
that directly or indirectly induced inflammatory
response. The damage of gut epithelial barrier may
facilitate the influx of endotoxin and other noxious
luminal content into the systemic. It may activate the
systemic inflammation and accelerate the
complication of AMS.
Acknowledgement
One of the authors A. A. is grateful to Department
of Science and Technology, India for the INSPIRE
Fellowship. Thanks are also to the Director, Defence
Institute of Physiology & Allied Sciences, Delhi - 54
for financial support.
References
1 Frisancho A R, Developmental functional adaptation to high
altitude: Review, Am J Hum Biol, 25 (2013) 151.
2 Zhou Q, Severe acute mountain sickness complicated by
multiple organ dysfunction syndrome, Emerg Med, 2 (2012)
113.
3 Dishari G, Kumar R & Pal K, Individual variation in
response to simulated hypoxic stress of rat, Indian J Exp
Biol, 50 (2012) 774.
4 Adak A, Maity C, Ghosh K, & Mondal K C, Alteration of
predominant gastrointestinal flora and oxidative damage of
large intestine under simulated hypobaric hypoxia,
Z Gastroenterol, 52 (2014) 180.
5 Adak A, Maity C, Ghosh K, Pati B R & Mondal K C,
Dynamics of predominant microbiota in the human
gastrointestinal tract and change in luminal enzymes and
immunoglobulin profile during high-altitude adaptation,
Folia Microbiol, 58 (2013) 523.
6 Zhou Q Q, Yang D Z, Luo Y J, Li S Z, Liu F Y & Wang G
S, Over-starvation aggravates intestinal injury and promotes
bacterial and endotoxin translocation under high-altitude
hypoxic environment, World J Gastroenterol, 17 (2011)
1584.
ADAK et al.: SMALL INTESTINAL HOMEOSTASIS & HYPOBARIC HYPOXIA
1105
7 Maity C, Adak A, Pathak T K, Pati B R & Mondal K C,
Study on the large intestinal cultivable microflora of rat
under varied environmental hyperbaric pressures,
J Microbiol Immunol Infect, 45 (2012) 281.
8 Miller G L, Use of dinitrosalicylic acid reagent for
determination of reducing sugar, Anal Chem, 31(1959) 426.
9 Brock F M, Cecil T T, Forsberg W & Buchanan-Smith J G,
Proteolytic activity of rumen microorganisms and effects of
proteinase inhibitors, Appl Environ Microbiol, 44 (1982)
556.
10 Yolton D P & Savage D C, Influence of certain indigenous
gastrointestinal microorganisms on duodenal alkaline
phosphatase in mice, Appl Environ Microbiol, 31
(1976) 880.
11 Kent T H, Fisher L J & Marr R, Glucuronidase activity in
intestinal contents of rat and man and relationship to
bacterial flora, Proc Soc Exp Biol Med, 140 (1972) 590.
12 Bradford M M, A rapid and sensitive for the quantitation of
microgram quantitites of protein utilizing the principle of
protein-dye binding, Anal Biochem, 72 (1976) 248.
13 Marklund S & Marklund G, Involvement of the superoxide
anion radical in autoxidation of pyrogallol as a convenient
assay for superoxide dismutase, Euro J Biochem, 47 (1974)
469.
14 Aebi H E, Catalase, in Methods in enzymatic analysis edited
by H. U. Bergmeyer, Verlag Chemie, Weinhiem, Germany
1983, 278.
15 Griffith O W, Determination of glutathione and glutathione
disulfide using glutathione reductase and 2-vinylpyridine,
Anal Biochem, 106 (1980) 207.
16 Anderson M E, Determination of glutathione and glutathione
disulfide in biological samples, Methods Enzymol,
113 (1985) 548.
17 Beuge J A & Aust S D, Microsomal lipid peroxidation,
Metab Enzmol, 52 (1978) 302.
18 Ganju L, Karan D & Srivastava K K, Induction of stress protein
in response to hypoxia, Indian J Exp Biol, 37 (1999) 344.
19 Rhee S H, Pothoulakis C & Mayer E A, Principles and
clinical implications of the brain-gut-enteric microbiota axis,
Nat Rev Gastroenterol Hepatol, 6 (2009) 306.
20 Marteyn B, Scorza F B, Sansonetti P J & Tang C, Breathing
life into pathogens: the influence of oxygen on bacterial
virulence and host responses in the gastrointestinal tract,
Cell Microbiol, 13 (2011) 171.
21 Yoshimoto M, Sasaki M, Naraki N, Mohri M & Miki K,
Regulation of gastric motility at simulated high altitude in
conscious rats, J Appl Physiol, 97 (2004) 599.
22 Ley R E, Peterson D A & Gordon J I, Ecological and
evolutionary forces shaping microbial diversity in the human
intestine, Cell, 124 (2006) 837.
23 Quigley E M & Quera R, Small intestinal bacterial
overgrowth: roles of antibiotics, prebiotics, and probiotics,
Gastroenterol, 130 (2006) S78.
24 Hooper L V & Macpherson AJ, Immune adaptations that
maintain homeostasis with the intestinal microbiota,
Nat Rev Immunol, 10 (2010) 159.
25 Kumar A & Goyal R, Gabapentin attenuates acute hypoxic
stress-induced behavioral alterations and oxidative damage in
mice: Possible involvement of GABAergic mechanism,
Indian J Exp Biol, 46 (2008) 159.
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