Clinical and Research Innovations
Section Editors: Mayank Goyal, MD, FRCPC, and Michael Tymianski, MD, PhD
Deep-Tissue Oxygen Monitoring in the Brain
of Rabbits for Stroke Research
Nadeem Khan, PhD; Huagang Hou, MD; Clifford J. Eskey, MD; Karen Moodie, MSc;
Sangeeta Gohain, MSc; Gaixin Du, MSc; Sassan Hodge, PhD; William C. Culp, MD;
Periannan Kuppusamy, PhD; Harold M. Swartz, MD, PhD
T
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their limited capability to directly and repeatedly measure
brain pO2 in a minimally invasive manner has restricted their
effective application in ischemic stroke where monitoring
oxygen levels are crucial for the development, and optimization of novel strategies for clinical translation. We report
electron paramagnetic resonance (EPR) oximetry using
implantable resonators to monitor brain pO2 in rabbit with
the goal to develop and test novel strategies that can significantly reduce brain loss in ischemic stroke. EPR oximetry
using particulate probes, such as lithium phthalocyanine
crystals or its derivatives, have been used to study tissue pO2
in a wide range of experimental systems, including muscle,
heart, brain, kidney, and liver in rodents6–9 and is now being
developed for clinical applications.10,11 Despite the benefits
of EPR oximetry, the currently available hardware technology limits pO2 measurement to a depth of 1 cm at L-band
frequencies (1.2 GHz). This is largely because of nonresonant losses of the microwave energy in the tissue of interest.
The penetration of microwave energy can be increased up
to ≈7 cm by using lower frequencies but this decreases the
signal/noise (S/N) ratio of the EPR signal, thus compromising the accuracy of measurements.12 To resolve this problem, we have pioneered an innovative design of implantable
resonators for pO2 measurement at depths >1 cm (Figure 1).
We have implemented this approach to monitor tissue pO2
at 2 sites in each hemisphere of the rabbit brain simultaneously. Our overall goal is to optimize the outcome of
ischemic stroke for clinical translation. To the best of our
knowledge, this is the first report of monitoring brain pO2 in
multiple sites and at depths >1 cm from the skull in rabbit
by EPR oximetry.
he primary event in the ischemic stroke is a rapid decline
in the oxygen levels after the loss of blood flow in specific
areas of the brain. Subsequent pathological processes results
in a central core area of severely ischemic tissue surrounded
by a region of moderate ischemic tissue (penumbra) with a
preserved cellular metabolism. The outcome of an ischemic
stroke depends on the size of the infarct core and the potential
to salvage the cells in the penumbra, which is hypoperfused,
and therefore, at risk of infarction but still viable. Such viable
penumbral tissue can be rescued by quick interventions that
can increase oxygen levels or slow metabolism in the ischemic
area to minimize oxidative injury on reperfusion.
Several strategies have been investigated to rescue ischemic
tissue using experimental models, especially rodents, but
largely failed in subsequent clinical trials. The rabbit model
of ischemic stroke using embolic clot is a promising model
for developing effective strategies. This model first led to the
prediction of the clinical response of recombinant tissue-type
plasminogen activator to restore blood flow in patients.1 The
drug is currently recommended for administration within 3
hours for best outcomes and has also shown modest benefit
when administered within 4.5 to 6 hours of clinical onset.2
The rabbit model of embolic clot is now considered as a pertinent model for translational research by the Stroke Therapy
Academic Industry Roundtable recommendations.3
To rationally develop effectual therapies, it is important
to understand the effect of ischemic stroke on oxygen levels (partial pressure of oxygen [pO2]) in the regions directly
affected by the pathology, as well as contralateral regions of
the rabbit brain. The potential changes in tissue po2 of contralateral regions may provide crucial information on adaptive
response, if any, of the brain to counteract ischemic stroke.
Such research will greatly benefit from the availability of
oximetry techniques that can directly and repeatedly measure
tissue pO2 in several regions of the brain.
Several methods are currently available for the assessment of brain pO2, including oxygen electrodes.4,5 However,
Description of the Innovation
Implantable Resonator
The implantable resonator is assembled with thin nonmagnetic
copper wire (0.3-mm wire gauge) and consists of a coupling
Received December 4, 2014; final revision received December 4, 2014; accepted December 22, 2014.
From the Department of Radiology, EPR Center for the Study of Viable Systems, Geisel School of Medicine at Dartmouth, Hanover, NH (N.K., H.H.,
S.G., G.D., S.H., P.K., H.M.S.); Norris Cotton Cancer Center, Dartmouth-Hitchcock Medical Center, Lebanon, NH (N.K., H.H., S.G., P.K., H.M.S.);
Department of Radiology, Dartmouth-Hitchcock Medical Center, Lebanon, NH (C.J.E.); Center for Comparative Medicine and Research, Dartmouth
College, Hanover, NH (K.M.); and Department of Radiology, Interventional Radiology, University of Arkansas for Medical Sciences, Little Rock (W.C.C.).
Correspondence to Nadeem Khan, PhD, EPR Center for the Study of Viable Systems, Geisel School of Medicine at Dartmouth, 48 Lafayette St,
Lebanon, NH 03766. E-mail [email protected]
(Stroke. 2015;46:00-00. DOI: 10.1161/STROKEAHA.114.007324.)
© 2015 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.114.007324
1
2 Stroke March 2015
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Figure 1. A, Implantable resonator with 10- and 15-mm length of transmission line for pO2 measurement at different depths. B, Schematic
representation of a surface loop resonator coupled to the implantable resonator for pO2 measurement at 4 sites in a rabbit brain. C, Typical
electron paramagnetic resonance spectra acquired from the implantable resonator with sensor loops (SL1–SL4) perfused with N2, 5% O2,
and 21% O2 for the purpose of calibration. D, Change in line width (LW) with pO2 (calibration) of each sensor loop from left to right.
loop (12–16 mm diameter) at one end and a transmission line
with sensor loops (0.4–0.5 mm inner diameter) at the other
end (Figure 1A). The sensor loops (or tips) are loaded with 30
to 50 μg of lithium phthalocyanine (LiPc, oximetry probe)13
crystals. The length of the transmission lines defines the depth
and can be varied as needed for the experiment. The number
of sensor loops and the distance between them can also be
varied to measure pO2 at ≥1 sites in the brain of rabbits. The
entire resonator is coated with a gas permeable and biocompatible Teflon AF2400.14 The mean area of the oximetry probe
at the surface of each sensor loop is estimated to be 1.3 to 1.6
mm2; EPR oximetry, therefore, samples a region that includes
many capillary segments and provides average pO2 at the site
of sensor loop.8,15 Histological examination of the cerebral tissue in the rabbit euthanized 4 weeks after the placement of
implantable resonator did not show any obvious accumulation
of inflammatory cells or blood cells surrounding the sensor
loops. Similar results were also evident as early as 7 days after
the placement of implantable resonator with 6-mm length of
transmission lines in the brain of rats.16
Procedure for the Placement of Implantable
Resonator in the Brain of Rabbits
The surgical procedure for the placement of implantable resonator in the brain of rabbit was in strict accordance with the
National Institutes of Health Guide for the Care and Use of
Laboratory Animals and approved by the Institutional Animal
Care and Use Committee of Geisel School of Medicine at
Dartmouth. The head of the anesthetized rabbit was antiseptically treated with Betadine, and 70% alcohol scrubs. A small
incision (2–3 cm) was made on the skin and burr holes were
gently created by using 18-gauge needle on the skull at predefined coordinates (anterior–posterior from bregma, −2.0
mm; medial–lateral from midline, 4 and 8 mm on each hemisphere; dorsal–ventral from surface of skull, 15 and 10 mm
in each hemisphere). The sensor loops, SL1 and SL4, were
located in the parietal cortex, whereas SL2 and SL3 were
located in the subcortex (basal forebrain/internal capsule)
region of the brain. The position of the sensor loops can be
altered depending on the coordinates of the compromised tissue after ischemic stroke. The sensor loops were placed at the
desired depth and the coupling loop was placed on the skull
below the skin, to allow inductive coupling with the external
surface loop resonator of the EPR spectrometer (Figure 1B).
The incision on the skin was closed with nonabsorbable 3-0
nylon suture and the rabbit was monitored for recovery as per
the Institutional Animal Care and Use Committee protocol.
The repeated measurements of brain pO2 by EPR oximetry
was started 72 hours (day 3) after the placement of implantable resonator and the measurements were repeated for 4
weeks.
In Vivo EPR Oximetry
EPR oximetry requires one-time implantation of the oxygen
probes (LiPc or implantable resonator), but rest of the procedure for pO2 measurement is entirely noninvasive and can be
Khan et al Oximetry in Rabbits for Stroke Research 3
38±1.0°C (monitored via a rectal probe) by keeping the animal under warm air during the EPR measurements. The EPR
settings were as follows: incident microwave power, 0.4 to 1.2
mW; modulation frequency, 24 kHz; magnetic field center,
410 G; scan time, 10 s, scan range, 8 to 12 G, and modulation amplitude not exceeding one third of the line width. The
implantable resonator appears as a signal void in T1-weighted
MRI scans, which can be used to confirm their position in the
brain of rabbits.
Figure 2. Tissue pO2 in the rabbit brain at 2 sites in each hemisphere measured simultaneously by multisite electron paramagnetic resonance oximetry. The sensor loops (SL) 1 and SL4 were
at a depth of 10 mm; SL2 and SL3 were at a depth of 15 mm in
the left and right hemisphere, respectively. The brain pO2 measurements were repeated on days 3, 5, 7, 14, 21, and 28.
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repeated as desired.8,17–19 The basis of oximetry is the paramagnetic nature of oxygen, which broadens the EPR signal of the
probe in proportion to the amount of oxygen. EPR oximetry
has unique capabilities and advantages compared with other
techniques, such as (1) direct measurement of absolute pO2 in
the tissue of interest, (2) pO2 is quantified through a physical
interaction of oxygen with the probe (does not require oxygen
consumption), (3) pO2 measurements can be made continuously and repeatedly as desired, without a confounding influence of previous measurements, (4) The oxygen sensors are
metabolically inert and coated with Teflon, therefore, do not
perturb the tissue microenvironment, including oxygen content, and (5) there is no other technique available at present to
make repeated measurement of tissue pO2 without the need to
reintroduce the probe for each measurement.
A 1.2-GHz EPR spectrometer equipped with a surface loop
resonator and a set of gradient coil for multisite oximetry
was used for monitoring brain pO2 in the rabbit. The anesthetized rabbit (2.5% isoflurane in 30% O2) was positioned in
the EPR magnet and the external surface loop resonator was
gently placed over the head region. A magnetic field gradient
of 1.7 G/cm per ampere was used to separate the EPR spectra from each sensor loop for simultaneous multisite oximetry.20 The peak-to-peak line widths of the EPR spectra were
used to determine pO2 by using the calibration of implantable
resonator (Figure 1C and 1D). The rabbit was maintained at
Results
Brain pO2 was measured for 20 to 25 minutes on day 3 and
the measurements were repeated on days 5, 7, 14, 21, and 28
(Figure 2). The mean (SD) baseline pO2 at each site (SL1–SL4)
in the brain on day 3 was 39.2 (2.2), 41.6 (1.4), 41.3 (1.7), and
43.6 (2.0) mm Hg, respectively, and only a modest variation
was observed in the measurements repeated subsequently for
≤4 weeks. To mimic the ischemic situation with low levels
of oxygen and test the response of implantable resonator, the
breathing gas was switched to 15% O2 for 15 minutes and then
returned to 30% O2, (Figure 3A). To test the potential effect
of hyperoxia on brain pO2, the breathing gas was switched to
carbogen (95% O2+5% CO2) for 20 to 25 minutes and then
returned to 30% O2, (Figure 3B). These experiments were performed on days 7, 14, 21, and 28. The brain pO2 measured at
4 sites decreased by ≈30% from baseline in rabbit breathing
15% O2. However, brain pO2 measured at 4 sites increased
significantly by ≥75% during carbogen breathing. An exponential quadratic function of time was used to determine minimum pO2 (PO2min) attained during 15% O2 on each day and
the time to reach the PO2min (Tmin) (Figure 4). Similar analysis was used to determine maximal pO2 (PO2max) attained on
each day and the time to reach maximum pO2 (Tmax) during
carbogen inhalation. These analyses suggest that it took 10
to 14 minutes to reach a minimal or maximal pO2 when the
breathing gas was switched from 30% O2 to 15% O2 or carbogen, respectively. A similar time scale was noted for the brain
pO2 to return to the baseline level when the breathing gas was
switched from 15% O2 or carbogen to 30% O2. We anticipate
that such temporal pO2 information will be extremely useful
in designing hyperoxic therapies to modulate oxygen levels in
ischemic stroke.
Figure 3. Typical changes in tissue pO2 of the rabbit brain at 2 sites in each hemisphere during (A) 30% O2 (baseline), 15% O2, and return
to 30% O2 breathing, (B) 30% O2, carbogen and return to 30% O2 breathing. The experiment was repeated for 4 consecutive weeks.
4 Stroke March 2015
Figure 4. A, Baseline (base), minimum (min), and maximum (max) brain pO2 determined using exponential quadratic function in rabbit
breathing 30% O2, 15% O2, and carbogen, respectively. B, Time required to reach minimal pO2 on 15% O2 breathing (Tmin), maximal pO2
on carbogen breathing (Tmax), time required to return to baseline pO2 when the gas was switched from 15% O2 to 30% O2 (*Tbase), and time
required to return to baseline pO2 when the gas was switched from carbogen to 30% O2 (**Tbase) in the experiments repeated on days 7,
14, 21, and 28. The pO2 obtained from all the sensor loops were pooled on each day to obtain average brain pO2 for these analyses.
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Discussion
To fully comprehend the pathology of stroke and rationally
develop strategies to rescue ischemic tissue, there is an unmet
need to understand the complex temporal changes in tissue
pO2 that occur during the course of ischemic stroke, a capability that previously has not been available. The results highlight
the ability of EPR oximetry using implantable resonator for
pO2 measurements at 4 sites simultaneously in the brain of a
rabbit. The pO2 measurements can be repeated as desired. A
rapid decline in tissue pO2 during 15% O2 breathing potentially highlights the immediate changes in the oxygen levels
that may occur in ischemic stroke. The extent of increase in
tissue pO2 during carbogen breathing is encouraging and can
be potentially used as a therapeutic strategy to improve oxygen levels and thus save vital tissue loss in ischemic stroke.
For the multisite oximetry approach, the position of sensor
loops should be carefully selected so that they are located in the
region of interest, that is, infarct core and penumbra after ischemic stroke. The design of the implantable resonator, including
the length of transmission lines, number, and distance between
the sensor loops can be modified as needed for a particular
experiment. A stable brain pO2 was observed from day 3, which
suggest that the experiments to investigate ischemic stroke can
be initiated as early as 3 days after the placement of implantable
resonator in the brain of rabbits. The measurement of tissue pO2
in the nonischemic contralateral brain can be used as internal
control and investigate adaptive response of the brain to ischemic stroke. The brain pO2 data presented here was obtained
in a rabbit to illustrate the capability of temporal monitoring by
EPR oximetry. We are currently implementing this technique
to investigate temporal changes in the brain pO2 in additional
rabbits during hyperoxia and ischemic stroke.
Conclusions
We have demonstrated a direct and longitudinal measurement
of absolute tissue pO2 at several sites simultaneously in the
brain of rabbit by EPR oximetry using implantable resonators, a capability, which was not available hitherto. Dynamic
information of cerebral pO2 can be used to test and to optimize strategies for improving treatment outcome of ischemic
stroke. EPR oximetry with implantable resonators should
also be useful to investigate the effect of other pathologies,
such as traumatic brain injury and cold injury, on the oxygen
levels in the brain of clinically pertinent animal models.
Sources of Funding
National Institutes of Health grants R21NS082585 to Dr Khan,
R01EB004031 to Dr Kuppusamy, and the Electron Paramagnetic
Resonance Center, Geisel School of Medicine at Dartmouth,
Lebanon, NH.
Disclosures
None.
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Key Words: carbogen ◼ EPR oximetry ◼ hyperoxia ◼ implantable resonator
◼ ischemia ◼ po2 ◼ stroke
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Deep-Tissue Oxygen Monitoring in the Brain of Rabbits for Stroke Research
Nadeem Khan, Huagang Hou, Clifford J. Eskey, Karen Moodie, Sangeeta Gohain, Gaixin Du,
Sassan Hodge, William C. Culp, Periannan Kuppusamy and Harold M. Swartz
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Stroke. published online January 22, 2015;
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