Direct Detection of Tissue Nitric Oxide in Septic Mice
Philip E.James, Ke J.Liu, Harold M.Swartz
EPR Center for the Study of Viable Biological Systems, Department of
Diagnostic Radiology,
Dartmouth Medical School, Hanover, NH 03755, USA
BACKGROUND: The role of nitric oxide (NO) in causing much of the pathophysiological events
observed in sepsis (tissue hypoxia, re-distribution of blood flow,
inhibition of mitochondrial oxygen
consumption) has been the focus of many research studies. Previously, we
have demonstrated the
importance of NO in maintaining normal blood flow and tissue pO2 in the
kidney, and changes in
kidney tissue oxygenation following treatment with endotoxin. Detection of
NO in vivo, however,
has until now been based on indirect measurement of by-products in blood. We
describe in this
paper the direct detection and, for the first time, a time course of nitric
oxide production in tissue
during an induced septic episode by an electron paramagnetic resonance (EPR)
spin-trapping
technique.
METHODS: Experimental sepsis was induced by injecting endotoxin
(3mg/25g body weight) into
the tail vein of mice. At various time points, diethyldithiocarbamate (DETC)
was injected
intraperitoneally to trap NO in tissue. The mice were placed into the core
of a "whole body", loopgap
resonator linked to an L-band EPR spectrometer, operating at 1.1 GHz. EPR
spectra were
recorded every minute over the following 60 minutes, and the growth in EPR
signal arising from NO
trapped as NO-Fe-(DETC)2 in the liver or kidney monitored. Approximately 30
minutes following
injection of DETC spin trap, an equilibrium was established between
formation of the NO-Fe-
(DETC)2 in the tissue, and removal of this complex from the circulation by
the kidneys. The
maximum EPR signal intensity recorded at this equilibration point reflected
levels of tissue NO
present at that time.
RESULTS AND CONCLUSIONS: Relative signal intensity arising from
NO-(DETC)2-Fe
measured directly from the liver and kidney of mice given endotoxin was
maximal at 6 hours post
endotoxin. We failed to detect an EPR signal from mice given pyrogen-free
saline. The quality of the
EPR signal obtained (high signal to noise ratio of 15:1) using this
experimental set-up and L-band
EPR hardware was such that we calculated the sensitivity to be in the
micromolar NO concentration
range. This enabled us to establish a time course of NO production in tissue
following endotoxin, but
also measurement of NO from other organs (kidney and spleen). Our EPR
results correlated well
with previous measurements of the concentration of total nitrite in the
blood of mice following
endotoxin, and probably reflect NO arising from inducible NO-synthase
enzymes as a result of
endotoxin stimulation.
This technique was extended to experiments in which we first implanted an
oxygen sensitive
material (gloxy) into the liver of mice, and then monitored NO production
following endotoxin. Due
to the fact that the EPR spectrum from gloxy and that of NO-Fe-(DETC)2 do
not overlap, we were
able to monitor NO production and pO2 simultaneously in tissue, in real
time. These techniques will
be of particular use in studying many other conditions in which NO effects
on tissue perfusion
and/or enhanced production of NO are implicated.
This work was presented at the 25th annual meeting of the
International Society of Oxygen
Transport to Tissue, and a paper published in the accompanying publication:
P.E. James, K.J. Liu,
and H.M. Swartz, “Direct Detection of Tissue Nitric Oxide in Septic Mice,”
Adv. Exp. Med. Biol. 454:181-187 (1998).
This work was presented at the 25th annual meeting of the
International Society of Oxygen
Transport to Tissue, and a paper published in the accompanying publication:
P.E. James, K.J. Liu,
and H.M. Swartz, “Direct Detection of Tissue Nitric Oxide in Septic Mice,”
Adv. Exp. Med. Biol. 454:181-187 (1998).