Nitric Oxide Levels in the Intestines of Mice Submitted to Ischemia
and Reperfusion: L-Arginine Effects
A.E.S.U. Cintra, J.L. Martins, F.R.S. Patrı´cio, E.M.S. Higa, and E.F.S. Montero
ABSTRACT
Objective. Usualy an experimental necrotizing enterocolitis experimental model, we
Investigated nitric oxide levels in intestinal tissues of newborn mice with or without
L-arginine therapy during sessions of ischemia and reoxygenation.
Methods. Twenty-six newborn mice from the Wistar EPM-1 lineage, weighing from 4.5
to 6.2 g, were randomly assigned to three groups: G-I/R, hypoxia and reoxygenation;
G-Arg, L-arginine treatment I/R; and G-CTL, controls. G-I/R and G-Arg mice underwent
twice a day during their first 3 days of life exposure to gas chambers with 100% CO
2
for
5 minutes at 22°C before reoxygenation with 100% O
2
for another 5 minutes. After 12
hours, all animals were sedated, laparotomized, and had samples of ileum and colon taken
and- either formalin fixed histopathologic examations or frozen to -80°C for estimation of
tissue nitric oxide levels. Intestinal injuries were classified according to the criteria of Chiu
et al.
Results. The G-I/R and G-Arg groups showed injuries characteristic of necrotizing
enterocolitis (NEC) with an improved structural preservation rate in G-Arg. The
concentration of nitric oxide in the Ileum was much higher with G-Arg (16.5 4.9; P =
0.0019) G-I/R (7.3 2.0). This effect was not observed in the colon: G-I/R = 10.7 4.6
versus G-Arg = 15.5 8.7 (P = .2480).
Conclusion. Supply of L-arginine increased tissue levels of nitricoxide and reduced
morphologic intestinal injury among mice undergoing I/R.
N
ECROTIZING ENTEROCOLITIS (NEC) is the
most common gastrointestinal disease in newborns
with high rates registered in the neonatal ITU. There is an
high incidence among premature mice with weights below
average (2% to 7%), yielding a high mortality rate (45%).
1,2
Its etiopathogenesis is not completely recognized; neverthe-
less prematurity, neonatal asphyxiation, immunologic im-
maturity, intestinal ischemia, pathogenic bacterial coloniza-
tion, and excess proteins in feedings seem to contribute to
the development of NEC.
3,4
Hypoxia produces a drastic reduction in production of
high-energy molecules.
5
Once the existing ATP is con-
sumed, failure of the ionic pump is observed, along with
passive diffusion of ions through cell membranes. The
accumulation of calcium inside the cell activates calpain
proteases; xanthine-dehydrogenase is changed into xan-
thine oxygenase (XO). During this phase, inflammatory
mediators, such as cytokines, leukotrienes, tumor necrosis,
and plateled aggregation factors appear. During reoxygen-
ation, accumulated hypoxanthine is oxidized into xanthine
and uric acid through XO; the freed free radicals—super-
oxide, hydrogen peroxide— are aggressive toward cell
membranes igniting lipid peroxidation. NADPH-oxidase
also produces free oxygen radicals.
Nitric oxide (NO) is the “relaxing factor deriving from
the endothelium” (EDRF). In a water solution under
physiologic conditions of oxygen tension and temperature,
it is an unstable gas product with an average life span of 33
to 50 seconds.
6,7
It acts momentarily as a vaso or dilat
reacting instantly with oxygen forming NO
2
, which in
solution generates nitrites (NO
2
-
) and nitrates (NO
4
-
).
From the Surgery and Experimentation Pos-Graduation Pro-
gram, Pediatric Surgery Division, Universidade Federal de São
Paulo, UNIFESP, Brazil.
Address reprint requests to Alvaro E S U Cintra, MD, Rua Sgto
Alcides de Oliveira, 215, Maranduba, Ubatuba, São Paulo,
Brazil, Cep 11.680-000. E-mail: cintra@pratica.com.br
0041-1345/08/$–see front matter © 2008 by Elsevier Inc. All rights reserved.
doi:10.1016/j.transproceed.2008.02.044 360 Park Avenue South, New York, NY 10010-1710
830 Transplantation Proceedings, 40, 830 – 835 (2008)