Simultaneous Measurement of NO. and pO2 from Tissue by In
Vivo EPR
James, P.E., 1,2, Miyake, M. 2 and Swartz, H.M.2
1 Department of Cardiology, Wales Heart Research Institute, University of
Wales
College of Medicine, Heath Park, Cardiff CF4 4XN, Wales, U.K.
2 Department of Oral and Maxillofacial Surgery, Kagawa Medical University,
Japan
3 EPR Center for the Study of Viable Biological Systems, Dartmouth Medical
School, Hanover,
NH, 03755 USA
INTRODUCTION: Although nitric oxide (NO.) is a central mediator in
septic shock and
inflammation, direct detection of tissue NO. in vivo, until recently has
been difficult, and
techniques have relied on indirect measurement of by-products in blood or
invasive technology.
An accurate technique for the direct detection of NO., to determine where it
is produced and
where it has concentrations sufficient to play an important role, is
critical. Moreover, because a
principal physiological effect is to maintain vascular tone and blood flow,
the relationship
between NO. production and tissue oxygenation must be assessed in vivo in
order to understand
fully the physiological and pathophysiological processes in which NO. is
implicated.
METHODS: A technique was developed that utilized electron
paramagnetic resonance (EPR) to
measure NO. and pO2 directly, and non-invasively, from tissue in vivo.
Diethyldithiocarbamate
(DETC) was injected with iron so as to complex with NO. in the tissue. Gloxy
(an oxygensensitive
paramagnetic material) also was implanted into the tissue of interest (brain
or liver).
Because the signals arising from gloxy and NO-Fe-(DETC)2 did not overlap,
they could be
monitored and measured simultaneously in vivo. The gloxy was not responsive
to NO. or DETC.
As model systems we injected either SNP (an NO. donor) into animals and
monitored NO. and
pO2 simultaneously from brain, or endotoxin (lipopolysaccharide; LPS) was
injected in order to
induce a septic episode and NO. and pO2 measured from liver.
RESULTS: We observed an immediate increase in NO-Fe-(DETC)2 in brain
following injection of
SNP, with maxima occurring at 26+/-5 min. A similar effect was seen on brain
pO2, reaching
maximum level at 30 min. Baseline brain pO2 was 20.3+/-4.1 mmHg compared to
32.4+/-3.7
mmHg at 30 min after SNP injection (n=7 each; data represent mean+/-s.d.).
We failed to detect
NO-Fe-(DETC)2 in animals given saline alone. Peak pO2 levels were higher in
animals given
SNP, but given saline in place of DETC/Fe (36.7+/-5.4 mmHg). We also found
that pO2 values
always dropped beneath baseline (comparing with pre-injection values and
with animals given
saline only as a control for SNP treatment) before returning to normal.
We also investigated the relationship between liver pO2 and NO. during an
induced septic
episode. Endotoxin was administered i.p. (saline was administered to
controls), and
liver pO2 and NO. monitored simultaneously. We found that (a) pO2 values
were significantly
lower in endotoxic mice compared to saline controls at 300 min and decreased
further at 360 min
(n=8 each), (b) NO. levels in liver were maximal at 360 min, (c) animals
surviving the 360
min time point showed a recovery in liver pO2 and NO. levels toward normal
values.
DISCUSSION: In these model systems, we have compared measurements of
pO2 at a single tissue
location with average NO. from all points sampled by DETC/Fe. It is
important to note, however,
that any of the methods currently available for making measurements of pO2
by EPR oximetry
may be used for simultaneous measurements of NO.. These include i.v.
injection of nanoparticles
(to sample Kupffer cells) or injection of a slurry of fine particles
directly into the tissue of
interest (sampling pO2 over a larger area). The results offer a new direct
approach to
investigating the role played by NO. in affecting tissue pO2 in vivo. Our
results showed that in the
brain, elevated NO. resulted in increased tissue oxygenation, whereas in the
liver during sepsis,
decreased tissue oxygenation was associated with over-production of the NO.
This illustrates the
potential usefulness of this technique for studying other pathophysiological
situations in which
elevated NO. and/or effects on tissue oxygenation are implicated.
REFERENCE:
P.E. James, M. Miyake, and H.M. Swartz, "Simultaneous Measurement of NO and
pO2 from
Tissue by In Vivo EPR," Nitric Oxide: Biology and Chemistry. 3:292-301
(1999).