chloramine-t and Disease-Models--Animal

Although the cause of intestinal necrosis in diseases such as necrotizing enterocolitis (NEC) is multi-factorial, oxidants and bacteria likely are key factors. Many animal models of NEC generate histologic necrosis, but it is rare to see the gross necrosis that is observed in infants. Here, we present a novel model that produces full-thickness intestinal necrosis when chloramine-T, an oxidizing agent, is introduced directly into isolated intestinal loops. The aim of this study was to determine the role of bacteria in this model.. Three-week-old mice underwent isolated ileal loop construction by suture ligation, and either chloramine-T or saline was injected into the loop. Intestines were then returned to the abdominal cavity, and the incision was closed for 24 h, after which the intestinal loops were analyzed histologically and microbiologically. To determine if bacteria home to the site of injury, some mice had intracecal injection of luminescent Pseudomonas aeruginosa (PA). These mice were sacrificed 2, 6, 8, and 24 h later, and luminescent bacteria were localized via photon camera imaging.. No gross necrosis was observed in the saline-treated group, but 59% of the animals in the chloramine-T-treated group had necrosis (p<0.001). Relative bacterial species numbers were calculated for untreated control animals and those with saline-treated loops and chloramine-T-treated loops without and with necrosis. Lactobacillus was detected in 60% of the control animals but was absent in all animals that underwent surgery. Methicillin-sensitive Staphylococcus aureus, PA, and Enterococcus faecium were present only in animals that underwent loop construction; however, bacterial communities did not differ according to loop treatment or the presence or absence of necrosis. After intracecal injection of PA, bacteria homed to the loop and proximal bowel even though the loop was discontinuous with the remaining bowel.. Intraluminal chloramine-T causes full-thickness necrosis in three-week-old mice and is characterized by a loss of probiotic bacteria such as Lactobacillus. Necrotic loops of bowel become colonized rapidly with pathogenic bacteria by unconventional mechanisms. Oxidant stress and colonization by pathogenic bacteria may play important roles in intestinal necrosis across a wide spectrum of pathologic states, including NEC.

Topics: Animals; Cecum; Chloramines; Disease Models, Animal; Enterocolitis, Necrotizing; Fluorescent Dyes; Ileum; Intestinal Mucosa; Intestines; Lactobacillus; Male; Mice; Mice, Inbred C57BL; Necrosis; Oxidants; Pseudomonas aeruginosa; Tosyl Compounds

The validity of using chloramine-T as a model compound for mimicing oxidative stress was examined using human serum albumin (HSA) as a model. Important sites of oxidation were studied by mild treatment with chloramine-T and by mutating 34Cys for a serine (C34S).. High-performance liquid chromatography (HPLC) combined with fluorescence detection to confirm the validity of chloramine-T as an oxidizing agent was used. Oxidized amino acid residues were detected by reaction with 5,5'-dithiobis(2-nitro benzoic acid), digestion with cyanogen bromide, followed by capillary electrophoresis. Protein conformation was examined by spectroscopic techniques.. From the HPLC analysis of human serum, the validity of using chloramine-T as an oxidizing agent was confirmed. At low chloramine-T concentrations (CT0.1-HSA, CT1-HSA), 34Cys and Met residues were oxidized, at medium concentrations (CT10-HSA), the tryptophan residue also appeared to be oxidized, and at the highest concentration (CT50-HSA), the net charge of Site II of HSA was found to be more negative. The two highest levels of oxidation of HSA (CT10-HSA, CT50-HSA) resulted in conformational changes with an increased exposure of hydrophobic regions, decreased high-affinity bindings of warfarin and ketoprofen and a reduced esterase-like activity. The latter protein also has a shorter plasma half-life and an increased liver clearance.. We succeeded in imitating oxidative damage to HSA using chloramine-T and the findings show that Site II is more affected than Site I and 34Cys, when HSA is exposed to oxidative stress.

Topics: Animals; Chloramines; Disease Models, Animal; Humans; Mice; Mutation; Oxidative Stress; Serum Albumin; Tosyl Compounds

The histologic examination and planimetric evaluation of tissue slices has been so far the only available technique for studying the extent of experimental myocardial infarcts in long-term studies; however, this approach is rather time-consuming when the sample size is large. This study describes a new biochemical method for the quantitation of myocardial scarring, based on the determination of left ventricular hydroxyproline, and collagen content at the end of the healing process. Accordingly, several modifications were introduced into previously existing methods for the assay of hydroxyproline: our method allows a linear estimation of hydroxyproline in the range from 0.5 to 5 micrograms, with a precision of +/- 6.1% and a day-to-day variation of +/- 10.5%. The reliability of this approach for studying infarct size has been verified in rats with coronary artery occlusion divided into a control group and in a treated group receiving nitroglycerin. The animals were killed 21 days after coronary ligation and randomly assigned for infarct size evaluation either to the histologic or the collagen method. By histology infarct size averaged 30.6 +/- 4.8% (mean +/- S.E.M.) of left ventricle (LV) in control rats and 16.2 +/- 5.8% of LV in nitroglycerin-treated animals; by the proposed alternative method left ventricular collagen content was 26.8 +/- 1.0 micrograms/mg of dry weight in control rats and 19.5 +/- 1.2 micrograms/mg of dry weight in nitroglycerin-treated animals; infarct size calculated from collagen content by a simple formula, was 37.4 +/- 2.6% of LV and 22.3 +/- 2.6% of LV, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arteries; Chloramines; Collagen; Coronary Vessels; Disease Models, Animal; Heart Ventricles; Hydrogen-Ion Concentration; Hydroxyproline; Ligation; Male; Myocardial Infarction; Nitroglycerin; Rats; Rats, Inbred Strains; Tosyl Compounds

The objective of this study was to develop an animal model representative of chronic human alpha-1-proteinase inhibitor deficiency. Eight dogs were treated with a mild oxidizing agent, chloramine T, with varying regimens for 3--27 wk. The capacity of the serum to inhibit both trypsin and elastase was examined and found to respond differently. Although immunologically determined levels of protease inhibitor did not change, the ability of serum to inhibit elastase in an in vitro assay decreased in direct response to chloramine T treatment. The trypsin inhibitory capacity was less affected. Emphysemalike alterations in lung morphology were observable when histologic sections were evaluated both subjectively and objectively by mean linear intercept measurements. The data suggest that this model parallels the emphysema associated with the genetic alpha-1-proteinase inhibitor deficiency in man.

Topics: alpha 1-Antitrypsin; Animals; Chloramines; Disease Models, Animal; Disinfectants; Dogs; Lung; Male; Microscopy, Electron, Scanning; Pancreatic Elastase; Tosyl Compounds