The aim of this study was to evaluate protective effects of glycomacropeptide (GMP), a kappa casein-derived peptide, in experimentally induced endotoxemia or bacteremia in mice. The results showed that BALB/c mice, given intraperitoneally (i.p.) GMP, 24h before intravenous (i.v.) injection of a high dose of lipopolysaccharide (LPS) from Escherichia coli, strongly inhibited serum levels of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6), measured 2h later by bioassays. In addition, GMP, administered 24h before infection of CBA mice with a sublethal dose of E. coli, significantly lowered the number of bacterial cells in the spleen. The analysis of main blood cell types in mice pretreated 24h prior to infection with GMP revealed significant increase in the content of granulocytes and immature neutrophils. We, therefore, postulate, that induction of myelopoiesis by GMP may be a primary cause of the increased clearance of bacteria during the development of bacteremia in mice.
Nitric oxide (NO), depending on the amount, time and source of generation may exert both, protective and deleterious actions during endotoxic acute lung injury (ALI). Evaluation of the expression and localization of NOS isoforms in the lung of lipopolysaccharide (LPS) - treated rats may contribute to understanding the role of NO in pathogenesis of ALI. Tissue samples (lung, heart, liver, kidney and spleen) as well as peripheral blood polymorphonuclear cells (PMNs) were collected from control male Wistar rats and LPS - treated animals, 15, 30, 60, 120 and 180 min after LPS injection (2 mg kg-1 min-1 for 10 minutes, i.v.). Levels of NOS-2 and NOS-3 mRNA and protein in tissues and PMNs were estimated by RT-PCR, Northern blotting and Western blotting. Additionally, myeloperoxidase (MPO) activity in tissue samples was assayed. NOS-3 mRNA as well as protein were detected in lungs of control animals; pulmonary NOS-3 expression was not influenced by LPS. The induction of NOS-2 mRNA in rat lungs and in PMNs isolated from peripheral blood was observed 15 minutes after LPS challenge. In contrast, increase of NOS-2 mRNA in the heart, kidneys, liver and spleen was observed 2-3 hours after LPS injection. In all tissues rise in NOS-2 mRNA was followed after 1-2 hours by increase of NOS-2 protein. Importantly, progressive leukocyte sequestration in the lung parenchyma that started as early as 15 min after LPS injection was revealed only in the lungs; in other organs no significant changes in MPO activity were detected up to 180 min after LPS injection. In conclusion, infusion of LPS caused much more rapid expression of NOS-2 in lungs as compared to the heart, kidneys, liver and spleen. Early induction of NOS-2 may depend on the LPS-stimulated rapid neutrophil sequestration within lung vasculature and fast induction of NOS-2 in sequestrated neutrophils.
Various animals must cope with some specific extreme environmental conditions and, as a consequence, they developed extremely efficacious adaptive defence responses. The mechanisms of the specific defences are more clearly visible in some species than in humans. Therefore, animal models of the human defence mechanisms should be selected accordingly. The appropriate, well responding models may be regarded as "biological amplifiers". This review is focussed on examples of effective defence against: (i) parturitional asphyxia, which extends fertility of mammals; (ii) diving asphyxia, which extends access of food in aquatic birds and mammals; (iii) endotoxemia, which provides survival of the fittest in mammals showing top fertility; (iv) deep hypothermia, which enables hibernating mammals to arouse. Each of the defences needs close co-operation of the cardiovascular, respiratory and temperature regulatory systems. Underlined problems regarding a choice of appropriate experimental animal models should stimulate renaissance of comparative physiology.
Intravenous lipopolysaccharide (LPS) leads to acute lung injury (ALI) in rats. The purpose of this study was to examine the anti-inflammatory and antioxidant efficacy of ketamine, propofol, and ketofol in a rat model of ALI. We induced ALI in rats via intravenous injection of LPS (15 mg kg-1). The animals were randomly separated into five groups: control, LPS only, LPS + ketamine (10 mg·kg-1·h-1), LPS + propofol (10 mg·kg-1·h-1), LPS + ketofol (5 mg·kg-1·h-1 ketamine + 5 mg·kg-1·h-1 propofol). LPS resulted in an increase in the release of pro-inflammatory cytokines, mRNA expression related with inflammation, production of nitric oxide, and lipid peroxidation. Ketamine prevented the increase in markers of oxidative stress and inflammation mediators, both in plasma and lung tissue. Propofol decreased the levels of cytokines in plasma and lung tissue, whereas it had no effect on the IL-1-beta level in lung tissue. Ketamine downregulated mediators of lung tissue inflammation and reduced the level of circulating cytokines and protected lung tissue against lipid peroxidation. Ketofol decreased the level of TNF-α and IL-1β in plasma, as well as expression of cyclooxygenase-2 mRNA and the nitrate/nitrite level in lung tissue. The results of this investigation support the hypothesis that ketamine may be effective in preventing ALI.
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Nitric oxide (NO) may cause sepsis-induced impairment of hypoxic pulmonary vasoconstriction (HPV). Although NO exerts many of its actions by activating soluble guanylate cyclase (sGC), there are several cGC-independent mechanisms that may lead to NO-induced vasodilation during endotoxemia. We investigated the role of sGC for the regulation of HPV during lipopolysaccharide (LPS) induced endotoxemia using 1H-(1,2,4)oxadiazole(4,3-)quinoxaline-1-one (ODQ), a specific inhibitor of sGC, in isolated, perfused, and ventilated mouse lungs. Without ODQ, lungs from LPS-challenged mice constricted significantly less in response to hypoxia as compared to lungs from mice not treated with LPS (26 ± 27% vs. 134 ± 37%, respectively, p < 0.05). 20 mg/kg ODQ, but not 2 mg/kg or 10 mg/kg, restored the blunted HPV response in LPS-challenged mice as compared to mice not challenged with LPS (80±14 % vs. 98±21 %). ODQ had no effect on baseline perfusion pressures under normoxic conditions. Analysis of pulmonary vascular P-Q relationships suggested that the restoration of pulmonary vascular response to hypoxia by ODQ is associated with a restoration of pulmonary vascular properties during normoxia. Our data show in a murine model that specific inhibition of sGC may be a new approach to restore HPV during endotoxemia.
Ammonium salt of N-(dithiocarboxy)sarcosine (DTCS) chelated to ferrous salt was tested as an NO-metric spin trap at room temperature for ex vivo measurement of NO production in murine endotoxaemia. In a chemically defined in vitro model system EPR triplet signals of NO-Fe(DTCS)2 were observed for as long as 3 hours, only if samples were reduced with sodium dithionite. This procedure was not necessary for the ex vivo detection of NO in endotoxaemic liver homogenates at X-band or in the whole intact organs at S-band, whereas only a weak signal was observed in endotoxaemic lung. These results suggest that in endotoxaemia not only high level of NO, but also the redox properties of liver and lung might determine the formation of complexes of NO with a spin trap. Nevertheless, both S- and X-band EPR spectroscopy is suitable for NO-metry at room temperature using Fe(DTCS)2 as the spin trapping agent. In particular, S-band EPR spectroscopy enables the detection of NO production in a whole organ, such as murine liver.
The study was made on 17 stallions of Polish Primitive Horses divided into four groups: I - control group, II - subject to LPS Escherichia coli injection in the dose of 0.05 μg/kg b.w., III - LPS in the dose of 0.1 μg/kg b.w. and IV - LPS in the dose of 0.3 μg/kg b.w. (n=5). The study was meant to determine the concentration of total protein (TPR), albumin (ALB) and aspartate aminotransferase (AspAT) in seminal plasma. Changes in the levels of all the above biochemical components in seminal plasma were noticed in stallions under endotoxin influence, especially after administration of the highest LPS dose. A statistically significant increase was noticed in TPR concentration (maximum in the 8th week), ALB concentration (maximum in the 24th hour). Changes in AspAT activity was also found with a maximum increase in the 6th week after LPS E. coli administration. The results of these studies show that endotoxemia induces changes in the biochemical properties of the seminal plasma in stallions, especially with reference to enzyme activity.
Ferrous-diethyldithiocarbamate (Fe(DETC)2) chelate is a lipophilic spin trap developed for NO detection by electron paramagnetic resonance (EPR) spectroscopy. Using this spin trap we investigated the kinetics of NO production in endotoxaemia in rats induced by lipopolysaccharide (Escherichia coli, 10 mg/kg). The NO-Fe(DE- TC)2 complex was found to give a characteristic EPR signal, and the amplitude of the 3rd (high-field) component of its hyperfine splitting was used to monitor the level of NO. We found that in blood, kindey, liver, heart and lung NO production starts to increase as early as 2 h after LPS injection, reaches the maximum 6 h after LPS injection and then returns to basal level within further 12-18 h. Interestingly, in the eye bulb the maximum of NO production was detected 12 h after LPS, and the signal was still pronounced 24 h after LPS. In brief, the highly lipophilic exogenous spin trap, Fe(DETC)2 is well suited for assessment of NO production in endotoxaemia. We demonstrated that the kinetics of increased production of NO in endotoxaemic organs, with the notable exception of the eye, do not follow the known pattern of NOS-2 induction under those conditions. Accordingly, only in early endotoxaemia a high level of NO is detected, while in late endotoxaemia NO detectability is diminished most probably due to concomitant oxidant stress.
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Bacterial endotoxin (lipopolysaccharide, LPS), at high concentration is responsible for sepsis, and neonatal mortality, however low concentration of LPS protected the pancreas against acute damage. The aim of this study was to investigate the effect of exposition of suckling rats to LPS on the course of acute pancreatitis at adult age. Suckling rat (30-40g) received intraperitoneal (i.p.) injection of saline (control) or LPS from Escherichia coli or Salmonella typhi (5, 10 or 15 mg/kg-day ) during 5 consecutive days. Two months later these rats have been subjected to i.p. caerulein infusion (25 µg/kg) to produce caerulein-induced pancreatitis (CIP). The following parameters were tested: pancreatic weight and morphology, plasma amylase and lipase activities, interleukin 1ß (IL-1 ß), interleukin 6 (IL-6), and interleukin 10 (IL-10) plasma concentrations. Pancreatic concentration of superoxide dysmutase (SOD) and lipid peroxidation products; malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) have been also measured. Caerulein infusion produced CIP in all animals tested, that was confirmed by histological examination. In the rats, which have been subjected in the neonatal period of life to LPS at doses 10 or 15 mg/kg-day x 5 days, all manifestations of CIP have been reduced. In these animals acute inflammatory infiltration of pancreatic tissue and pancreatic cell vacuolization have been significantly diminished. Also pancreatic weight, plasma lipase and a-amylase activities, as well as plasma concentrations of IL-1ß and IL-6 have been markedly decreased, whereas plasma anti-inflammatory IL-10 concentration was significantly increased in these animals as compared to the control rats, subjected in the infancy to saline injection instead of LPS. Caerulein-induced fall in pancreatic SOD concentration was reversed and accompanied by significant reduction of MDA + 4 HNE in the pancreatic tissue. The effects of LPS derived from E.coli or S.typhi were similar. Pretreatment of suckling rats with LPS at dose of 10 mg/kg-day x 5 days resulted in the most prominent attenuation of acute pancreatitis at adult age, whereas LPS at dose of 5 mg/kg-day x 5 days given to the neonatal rats failed to affect significantly acute pancreatitis induced in these animals 2 months later. We conclude that: 1/ Prolonged expositon of suckling rats to bacterial endotoxin attenuated acute pancreatitis induced in these animals at adult age. 2/ This effect could be related to the increased concentration of antioxidative enzyme SOD in the pancreatic tissue and to the modulation of cytokines production in these animals.
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