modulation of small intestinal homeostasis along with its...

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: [email protected]

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


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



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.


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



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]


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]


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.

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