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The Regulation of Brain Tissue Oxygenation - An In Vivo EPR Study

Jeffrey F. Dunn1, Jennifer Merlis1, Michelle A. Abajian1, Stalina A. Grinberg2,
Eugene Demidenko3, Huagang Hou2, and Oleg Y. Grinberg2 

1Department of Diagnostic Radiology, NMR Center, Dartmouth Medical School, Hanover, NH 03755 USA;
 2Department of Diagnostic Radiology, EPR Center, Dartmouth Medical School, Hanover, NH 03755, USA;
 3Section of Biostatistics and Epidemiology, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756, USA

 

INTRODUCTION

Oxygen is a key metabolite for brain function, and so it is important to understand the regulatory mechanisms that determine oxygen
delivery and set the levels of oxygen in the brain. Changes in tissue PtO2 with acute changes in arterial PtO2 have been shown in brain,
using oxygen electrodes and fluorescence quenching methods. These methods, however, require acute insertion of the measuring
device and therefore usually require anesthesia and produce local trauma, both factors that can perturb the PtO2. We have developed
a method to measure brain PtO2 non–invasively in awake restrained animals using EPR oximetry. In the current study we used
EPR oximetry to test the hypothesis that exposure to hypoxia will induce adaptations that will lead to a normalization of PtO2
 in the brain to pre-hypoxic levels.

MATERIALS AND METHODS

Precalibrated lithium phthalocyanine (LiPc) crystals were used as the oxygen sensitive material. LiPc was implanted into the frontal
cortex of 150-200g Wistar rats using a 25G needle under ketamine/xylazine anesthesia (100mg/10mg/kg), 4-6 days before EPR measurements.
Awake animals were restrained in a conical plastic bag (DecapiCone, Braintree Scientific, MA) and placed in the machine with the surface-loop
resonator positioned above the LiPc implant. The PtO2 of each animal was calculated from the linewidths of 3-4 EPR spectra. Inspired gas
(purchased premixed) was delivered at 2 L/min though Tygon tubing to the tip of the bag. Rats were acclimated to ½ atm of hypobaric hypoxia
in 40 gallon drums for 36 days. The chambers were returned to normobaric pressures daily for approximately one hour for cage cleaning.
Ambient temperature, weight, and pressure were recorded daily. EPR oximetry was conducted while the animals breathed 21% followed
by 10% O2. Rats were measured on 3 days before acclimation and 3 days near the end of the acclimation period (over days 27-36).

In each animal, before acclimation and then near the end of the acclimation period (over days 27-36), PtO2 was measured on three successive
days with the animal breathing 21% O2 followed by 10% O2.

On these dates, upon returning to ambient pressure, animals were maintained at 10% O2 to simulate the inspired oxygen of their acclimation
conditions. Since 10% O2 was now their adapted condition, we reversed the order of gas delivery during EPR oximetry such that these
animals were first exposed to 10% O2 followed by 21% O2. The acute response to hypoxia also was measured with 4 unacclimated rats.
PTO2 was measured when breathing 21% O2, followed by 10% O2, followed by 10%O2 with 10% CO2 (all balance N2).

 

RESULTS

Using ANOVA, it was determined that the values of PtO2 measured on the 3 pre-acclimation dates were not significantly different
and so the data were grouped. The same was true for the data collected at the end of acclimation. In pre-acclimated rats, when the inspired gas
was reduced from 21% to 10%, the PtO2 declined from 26.7±2.2 to 13.0±1.5 mm Hg (mean±SD, n=4), Figure 1. The addition of 10% CO2 to
the hypoxic breathing gas resulted in the PtO2 returning to the levels measured when breathing 21% O2. The PtO2 increased significantly
with acclimation (Figure 2). The average PtO2 in the brain before acclimation began was 17.9±3.3 mm Hg (mean±SD, n=6) while breathing 21% O2
and 8.3±1.9 mm Hg while breathing 10% O2. At the end of acclimation, animals breathing 21% O2 had a PtO2 of 32.8±8.0 mm Hg,and a PtO2
of 16.7±2.9 mm Hg while breathing 10% O2. The average decline in PtO2 for a 50% reduction in inspired O2 was 54% before acclimation and 49%
at the end of acclimation. The PtO2 was not significantly different between the pre-acclimation values measured while breathing 21% O2,
and the end of acclimation values measured while breathing 10% O2.

 














Figure 1. Brain PtO2 during acute exposure to hypoxia and hypoxia+hypercapnia. (mean±SD, n=4).
There was no significant difference between the PtO2 measured while breathing 21% O2,
 and that measured while breathing 10% O2 with 10% CO2 (ANOVA, p<0.05).

 

Since most methods for measuring PtO2 are invasive, the tissue is subject to acute trauma and the subject needs to be anesthetized. |
Once the oxygen sensitive material is implanted, EPR oximetry can provide repeated non-invasive measurements of the PtO2
at the implanted sites within tissue in vivo. Histological examination shows no inflammation and indicates that the implant is in good
contact with the surrounding tissue.

 















Figure 2.  Average brain PtO2 values during inhalation of 21% and 10% O2 before and after acclimation to hypoxia.
Data are combined from measurements on 3 separate dates before acclimation and 3 dates ranging over
27 to 36 days of acclimation to hypoxia( mean±SD, n=6). *denotes significant difference from respective pre-acclimation values.

 

DISCUSSION

This study confirms, in awake animals, that PtO2 will change with an acute change in inspired PtO2. The PtO2 declined by approximately 50%
for a 50% reduction in FiO2. Since hypoxia can stimulate CBF under extreme conditions, we determined if there was sufficient reserve of CBF
to maintain PtO2. We exposed animals to 21% O2, then 10% O2, observed the decline in PtO2, and then supplemented the 10% O2 with 10% CO2.
The CO2 stimulates CBF and is not expected to change CMRO2. The PtO2 increased to within the “normoxic” range observed when
breathing 21% O2, even though the inspired O2 remained at 10%, indicating that there was sufficient reserve of CBF to maintain PtO2.

It was shown the maximal change in PtO2 was achieved after 7 days for animals acclimated to ½ atm while breathing 21% O2. As with the first study,
there was a significant increase in the brain PtO2. Throughout the current study, the acute reduction of inspired O2 from 21% to 10% resulted in,
approximately, a 50% reduction of PtO2. A key observation in this study is the fact that when data are compared between the pre-acclimation group
breathing 21% O2 and the end of acclimation group breathing 10% O2, the PtO2 is unchanged. This evidence of regulation of PtO2 is consistent
with the existence of a pre-determined set-point around which brain PtO2 is regulated under conditions of chronic exposure. If this is true,
then after exposure to chronic hypoxia for a time sufficient to allow for adaptation, which may be as quickly as 7 days, the PtO2 in brain will return
to the pre-hypoxia value.

 

SUMMARY AND CONCLUSIONS

PtO2 in acclimated animals was similar when breathing 10% O2, as when breathing 21% O2 before acclimation.  Maintenance of PtO2 after
exposure to chronic hypoxia is consistent with a regulatory mechanism that senses PtO2. There are adaptive mechanisms initiated by hypoxia,
which result in acclimation to chronic hypoxia. The brain adapts to chronic hypoxia by returning the tissue to a pre-hypoxic PtO2, indicating that
there are mechanisms (such oxygen sensitive HIF-1a stabilization system) that are capable of sensing PtO2 and of initiating a cascade of events,
which result in the normalization of PtO2. These data support the concept that CBF is not regulated by a PtO2 sensor at this level of acute hypoxia.

 

ACKNOWLEDGEMENTS

This work was supported by NIH grant “Near Infrared/MR System for Imaging Brain Oxygenation”, RO1 EB002085 and used the facilities
of NIH (NIBIB) P41 EB002032, “EPR Center for the Study of Viable Systems.”

 

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