Alteration of Predominant Gastrointestinal Flora and Oxidative Damage of Large Intestine Under Simulated Hypobaric Hypoxia

Abstract

Hypobaric hypoxia is an immediate and crucial starting mechanism of acute mountain sickness included with some non-specific gastrointestinal (GI) complications. To study the effect of hypoxia on GI microflora and its upshot to this system, male albino rats were exposed to 55 kPa (air pressure ~ 4872.9 m altitude) consecutively 30 days for 8 hours/day. The different indicator group of large intestinal microbial populations were enumerated and correlated with the levels of antioxidant indicators like catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), reduced glutathione (GSH) and oxidized glutathione (GSSG) of large intestinal epithelial cells. In addition, the histological study was performed by haematoxylin eosin (HE), periodic acid schiff staining (PAS) and scanning electron microscopy (SEM). It was observed that the density of total aerobes (104 folds) significantly (p < 0.05) decreased but the population of total anaerobes (209 folds) and Escherichia coli (125 folds) elevated after 30 days of hypoxic stress. The strict anaerobes like Bifidobacterium spp. (3 folds), Bacteroides spp. (134 folds), Lactobacillus spp. (7 folds) and other selected obligate anaerobes like Clostridium perfringens (40 folds), Peptostreptococcus spp. (21 folds) increased in respect to their control population. The growth direction index (GDI) of anaerobic populations was positive and correlated with gas formation aptitude. The activities of CAT and SOD in the large intestinal epithelia decreased significantly (p < 0.05) and GSH/GSSG pool turned into oxidized state with higher MDA (p < 0.05) formation. Histological study revealed the necrotized epithelial layer with higher lymphocytes infiltration in lamina propia accompanied by reduction of acidic mucins secreting goblet cells. From this experiment, it can be hypothesized that high altitude induced hypoxia manipulated the bacterial imprint and damaged the epithelial barrier of the large intestine which may cause systemic infection.

Zusammenfassung

Hypobare Hypoxie ist ein unmittelbar einsetzender und kritischer Startmechanismus der akuten Bergkrankheit, der mit einigen unspezifischen gastrointestinalen Komplikationen einhergeht. Um die Wirkung von Hypoxie auf die Mikroflora des Gastrointestinaltrakts und deren systemische Auswirkung zu untersuchen, wurden männliche Albinoratten an 30 aufeinanderfolgenden Tagen 8 Stunden pro Tag einem Druck von 55 kPa (Luftdruck entspricht 4872,9 Höhenmeter) ausgesetzt. Die unterschiedlichen populationsbezogenen Indikatoren der Dickdarmflora wurden quantifiziert und mit den Spiegeln der Antioxidant-Indikatoren wie Katalase (CAT), Superoxiddismutase (SOD), Malondialdehyd (MDA), reduziertem Glutathion (GSH) und oxidiertem Glutathion (GSSG) der Epithelzellen des Dickdarms korreliert. Zusätzlich wurde eine histologische Untersuchung mit Hämatoxylin-Eosin-Färbung (HE-Färbung), Perjodsäure-Schiff-Reaktion (PAS-Reaktion) und Rasterelektronen­mikroskopie durchgeführt. Die Untersuchungen ergaben, dass die Dichte aller Aeroben (104-fach) signifikant (p < 0,05) abnahm, während die Population aller Anaeroben (209-fach) und von Escherichia coli (125-fach) nach 30 Tagen hypoxischer Belastung zunahm. Strikte Anaerobe wie Bifidobacterium spp. (3-fach), Bacteroides spp. (134-fach), Lactobacillus spp. (7-fach) und andere ausgewählte obligate Anaerobe wie Clostridium perfringens (40 fach) und Peptostreptococcus spp.  (21-fach) vermehrten sich im Vergleich zu ihrer Kontrollpopulation. Der Wachstumsindex (growth direction index GDI) der anaeroben Population war positiv und korrelierte mit der Fähigkeit zur Gasbildung. Die Aktivität von CAT und SOD in den Epithelzellen des Dickdarms nahm signifikant ab (p < 0,05) und der GSH/GSSG Pool ging mit einer erhöhten MDA (p < 0,05) Bildung in den oxidierten Zustand über. Die histologische Untersuchung zeigte eine nekrotisierte Epithelschicht mit einer erhöhten Lymphozyten-Infiltration in der Lamina Propia, begleitet von einer Reduktion an saurem Mucin-bildenden Becherzellen. Dieser Versuch legt die These nahe, dass eine durch große Höhe verursachte Hypoxie die bakterielle Flora verändert und die Epithelbarriere des Dickdarms schädigt, was zu einer systemischen Infektion führen kann.

Key words

hypobaric hypoxia - acute mountain sickness - large intestine - histological study - systemic infection

Schlüsselwörter

hypobare Hypoxie - akute Bergkrankheit - Dickdarm - histologische Untersuchung - systemische Infektion

• References

• 1 Hooper LV, Macpherson AJ. Immune adaptations that maintain homeostasis with the intestinal microbiota. Nat Rev Immunol 2010; 10: 159-169
• 2 Ley RE, Peterson DA, Gordon JI. Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 2006; 124: 837-848
• 3 Possemiers S, Grootaert C, Vermeiren J et al. The intestinal environment in health and disease: recent insights on the potential of intestinal bacteria to influence human health. Curr Pharm Des 2009; 15: 2051-2065
• 4 Westerterp KR, Kayser B, Brouns F et al. Energy expenditure climbing Mt. Everest. J Appl Physiol 1992; 73: 1815-1819
• 5 Camp JG, Kanther M, Semova I et al. Patterns and scales in gastrointestinal microbial ecology. Gastroenterology 2009; 136: 1989-2002
• 6 Butterfield GE, Gates J, Fleming S et al. Increased energy intake minimizes weight loss in men at high altitude. J Appl Physiol 1992; 72: 1741-1748
• 7 Westerterp KR, Kayser B, Brouns F et al. Energy expenditure climbing Mt. Everest. J Appl Physiol 1992; 73: 1815-1819
• 8 Anand AC, Sashindran VK, Mohan L. Gastrointestinal problems at high altitude. Trop Gastroenterol 2006; 27: 147-153
• 9 Diniz YS, Faine LA, Galhardi CM et al. Monosodium glutamate in standard and high-fiber diets: metabolic syndrome and oxidative stress in rats. Nutrition 2005; 21: 749-755
• 10 Maity C, Adak A, Pathak TK et al. Study on the large intestinal cultivable microflora of rat under varied environmental hyperbaric pressures. J Microbiol Immunol Infect 2012; 45: 281-286
• 11 Aebi HE. Catalase. Methods in enzymatic analysis. Weinhiem, Germany: Verlag Chemie; 1983: 278-282
• 12 Marklund S, Marklund G. Involvement of the superoxide anion radical in autoxidation of pyrogallol as a convenient assay for superoxide dismutase. Eur J Biochem 1974; 47: 469-474
• 13 Beuge JA, Aust SD. Microsomal lipid peroxidation. Metab Enzmol 1978; 52: 302-310
• 14 Griffith OW. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 1980; 106: 207-212
• 15 Anderson ME. Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol 1985; 113: 548-555
• 16 Ojano-Dirain C, Iqbal M, Wing T et al. Glutathione and respiratory chain complex activity in duodenalmitochondria of broilers with low and high feed efficiency. Poult Sci 2005; 84: 782-788
• 17 Murata H, Yagi M, Kubota M et al. Embryonic development of the myenteric nerve plexus in the rat small intestine as revealed by light and scanning electron microscopy. Arch Histol Cytol 2003; 66: 453-468
• 18 Dati F, Schumann G, Thomas L et al. Interim reference ranges for 14 serum proteins based on standardization with CRM 470. Eur J Clin Chem Clin Biochem 1996; 34: 517-520
• 19 Deplancke B, Gaskins HR. Microbial modulation of innate defense: goblet cells and the intestinal mucus layer. Am J Clin Nutr 2001; 73: 1131S-1141S
• 20 Gombošov L, Lazúrová I, Zakuciová M et al. Genes of intestinal Escherichia coli and their relation to the inflammatory activity in patients with ulcerative colitis and Crohn’s disease. Folia Microbiol 2011; 56: 367-372
• 21 Yoshimoto M, Sasaki M, Naraki N et al. Regulation of gastric motility at simulated high altitude in conscious rats. J Appl Physiol 2004; 97: 599-604
• 22 Loiacono LA, David SS. Detection of hypoxia at the cellular level. Crit Care Clin 2010; 26: 409-412
• 23 Zhou QQ, Yang DZ, Luo YJ et al. Over-starvation aggravates intestinal injury and promotes bacterial and endotoxin translocation under high-altitude hypoxic environment. World J Gastroenterol 2011; 17: 1584-1593