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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2014 Feb 19;34(5):890–896. doi: 10.1038/jcbfm.2014.35

Tissue oxygen is reduced in white matter of spontaneously hypertensive-stroke prone rats: a longitudinal study with electron paramagnetic resonance

John Weaver 1,6, Fakhreya Y Jalal 2,3,6, Yi Yang 2, Jeffrey Thompson 2, Gary A Rosenberg 2,4,5, Ke J Liu 1,2,*
PMCID: PMC4013771  PMID: 24549186

Abstract

Small vessel disease is associated with white-matter (WM) magnetic resonance imaging (MRI) hyperintensities (WMHs) in patients with vascular cognitive impairment (VCI) and subsequent damage to the WM. Although WM is vulnerable to hypoxic-ischemic injury and O2 is critical in brain physiology, tissue O2 level in the WM has not been measured and explored in vivo. We hypothesized that spontaneously hypertensive stroke-prone rat (SHR/SP) fed a Japanese permissive diet (JPD) and subjected to unilateral carotid artery occlusion (UCAO), a model to study VCI, would lead to reduced tissue oxygen (pO2) in the deep WM. We tested this hypothesis by monitoring WM tissue pO2 using in vivo electron paramagnetic resonance (EPR) oximetry in SHR/SP rats over weeks before and after JPD/UCAO. The SHR/SP rats experienced an increase in WM pO2 from 9 to 12 weeks with a maximal 32% increase at week 12, followed by a dramatic decrease in WM pO2 to near hypoxic conditions during weeks 13 to 16 after JPD/UCAO. The decreased WM pO2 was accompanied with WM damage and hemorrhages surrounding microvessels. Our findings suggest that changes in WM pO2 may contribute to WM damage in SHR/SP rat model, and that EPR oximetry can monitor brain pO2 in the WM of small animals.

Keywords: brain oxygen, EPR oximetry, vascular cognitive impairment, white matter

Introduction

Brain cells poorly tolerate hypoxia, and even short periods of low oxygen (O2) can initiate molecular pathways that are lethal to cells in both the gray and white matter (WM). Deep WM is a common site of hypoxic/ischemic injury in the elderly where it is associated with cognitive decline, gait disturbances, and focal ischemia leading to vascular cognitive impairment (VCI). Magnetic resonance imaging (MRI) reveals white-matter hyperintensities (WMHs) in the elderly located in the periventricular and subcortical regions, and considered to be due to strokes and secondary small vessel disease. An alternative mechanism is that the deep WM is vulnerable to hypoxia for a variety of reasons. Studies in humans suggest that there is impairment in the ability of the deep WM to respond to increased demand by raising cerebral blood flow or O2.1 Although glutamate mediated excitotoxicity, inflammatory cytokines, and protease activation result in oligodendrocyte death in acute ischemic injury, the pathophysiology of WM injury in chronic vascular disease has not been fully explored.2 Small vessel disease is associated with WMHs in patients with VCI; damage to small vessels causes an inflammatory response with disruption in the blood–brain barrier (BBB) associated with expression of matrix metalloproteinases (MMPs) and subsequent damage to the WM. Vascular dementia patients show expression of MMPs in regions of loss of myelin.3, 4, 5 In several animal models of hypoxic hypoperfusion, damage to the WM has been associated with BBB disruption, breakdown of myelin basic protein and oligodendrocyte death.6 Spontaneously hypertensive stroke-prone rat (SHR/SP) is considered as the optimal experimental model for studying hypertensive damage leading to stroke and WM damage secondary related to hypertensive damage in the small vessel form of VCI observed in older patients.7, 8

Recently, we reported a novel model of WM injury in the SHR/SP fed the Japanese permissive diet (JPD), consisting of low protein and high salt, combined with permanent unilateral carotid artery occlusion (UCAO), which leads to extensive injury to the WM and death 4 weeks after starting the diet and occlusion. In addition to the WM damage that can be shown by MRI scans, there are associated cognitive deficits as shown with Morris water maze testing.8 A neuroinflammatory response associated with MMPs and tumor necrosis factor-α with BBB opening and demyelination was observed. Similarly in acute ischemia, there is elevation in MMPs associated with BBB leakage and knockout of MMP-9 reduces BBB disruption.9, 10, 11 Surprisingly, in light of the high significance of O2 in brain physiology, O2 measurement in the WM, and specifically related to VCI and WM injury has not been measured or explored in vivo although both conditions have been associated with stroke or transient ischemic attack and the diffuse pattern of injury is highly suggestive of a hypoxic injury.2, 7, 12, 13, 14

Direct measurement of O2 level in tissues as the partial pressure of oxygen (pO2) in the brain is one of the most important variables in many physiologic, pathologic, and therapeutic processes.15, 16, 17 The brain tissue is highly sensitive to changes in availability of O2 and pathological changes can occur if there are significant changes in the delivery or utilization of O2 in local regions of the brain.18, 19 Electron paramagnetic resonance (EPR) oximetry is a unique method that has several advantages compared with other O2 measurement methods, including the use of oxygen-sensitive electrodes or surface multielectrode arrays phosphorescence quenching, and near infrared spectroscopy. Electron paramagnetic resonance oximetry allows for repetitive and highly accurate measurement of localized tissue pO2;20 thus, it is a novel and innovative method for interstitial pO2 measurement in living animals and has proven to be an important tool in the study of brain tissue pO2.20, 21, 22, 23 Briefly, molecular O2, being paramagnetic, broadens the EPR spectral lines of other paramagnetic species, or paramagnetic probes.20 Measured changes in EPR spectral linewidth have been used to estimate O2 and recently, localized cerebral interstitial pO2 can be monitored and characterized at a single specific site in vivo using stable paramagnetic lithium phthalocyanine (LiPc) particles.21, 22, 23 These crystals have several desirable properties as probes for pO2 including high sensitivity, resistance to chemical reactions, and high degree of inertness in biologic systems.24 In addition, MRI can be used to determine the exact location of the crystal in tissue.24 These advancements have established EPR oximetry as a versatile method for minimally invasive, highly sensitive, and repetitive measurements of pO2 in the brain.

We hypothesized that SHR/SP with JPD/UCAO has reduced tissue oxygenation in the deep WM, and tested the hypothesis by measuring tissue pO2 over several months, using EPR with LiPc crystals implanted in the deep WM of three groups of SHR/SP: (1) with JPD/UCAO; (2) with sham carotid surgery; and (3) with no intervention. Tissue O2 was monitored from 6 to 16 weeks with the JPD/UCAO started in the twelfth week when the hypertension becomes significant and no strokes are present as previously reported.8 Herein, we showed the stability of the method by recording O2 in normotensive Wistar-Kyoto (WKY) rats from 12 to 16 weeks, and the sensitivity by showing a response in the normotensive rats to changes in respired oxygen. This is the first report to show hypoxia in the WM of SHR/SP with JPD/UCAO.

Materials and methods

Animal Preparation

All experimental protocols were reviewed and approved by the University of New Mexico Laboratory Animal Care Committee (Protocol #12-100779-HSC) and conformed to the National Institutes of Health Guidelines for use of animals in research. The experiments are reported here in accordance with the ARRIVE guidelines. Wistar-Kyoto male rats, 270 to 280 g, and SHR/SP male rats, 260 to 270 g, were obtained from Charles River Laboratory (Wilmington, MA, USA). Animals were housed in an AAALAC approved Animal Research Facility and were maintained in a climate-controlled vivarium with a 12-hour light–dark cycle and free access to food and water. In the earlier report, we showed that the SHR/SP showed an increase in blood pressure up to the twelfth week; without treatment the blood pressure remained at that elevated pressure and with JPD/UCAO there was a continued rise in blood pressure.8 Since the experiments were performed from 6 to 16 weeks of age, physiologic measurements other than blood pressure were not made.

For all surgical stereotaxic LiPc implantation and UCAO procedures, 4.0% isoflurane in N2O:O2 (70:30%) was used for anesthesia induction. Anesthesia was maintained with 2.0% isoflurane in N2O:O2 (70:30%) during all surgeries. Animals were anesthetized throughout the EPR measurements with 2.0% isoflurane in N2O:O2 (70:30%) after induction at 4.0% isoflurane in N2O:O2 (70:30%). Physiologic monitoring during all procedures comprises measurement and maintenance of core (rectal) temperature at 37.5±0.5°C using a heating pad or a heat lamp.

Implantation of Electron Paramagnetic Resonance Oximetry Probe Lithium Phthalocyanine and Unilateral Carotid Artery Occlusion Surgery

For every animal, correct assignment of the implantation site in the WM was determined using the Rat Brain Atlas25 and was confirmed by MRI and postmortem in the brain tissue slice. Lithium phthalocyanine was a gift from Dr Harold Swartz (EPR Center, Dartmouth College, NH, USA). Under anesthesia (2.0% isoflurane), a pinhole on the parietal skull was made at the stereotaxic position of AP: −3.1 mm and L: 1.8 mm right from the midline. A small LiPc crystal (approximate diameter 0.2 mm) was placed at a depth of −2.4 mm using a microdialysis guide cannula with an inner diameter of 0.24 mm (CMA microdialysis, Stockholm, Sweden). Rats were allowed to recover from implantation 48 to 72 hours before further study. The implantation procedure is well characterized and under our experimental conditions, inflammation, tissue damage or reaction to LiPc or the implantation procedure was not observed as previously described.21, 22, 23, 24, 26 For UCAO, at 12 weeks of age, male SHR/SP with LiPc implants was anesthetized with 2.0% isoflurane and surgery was induced. Briefly, under a surgical microscope, a ventral midline incision was made to expose the right carotid artery. After isolation, the right carotid artery was double ligated permanently with 4-0 silk sutures. After UCAO, rats were placed on a JPD (JPD/UCAO) (16% protein, 0.75% potassium, 4% sodium; Ziegler Bros, Inc., Gardner, PA, USA) with 1% sodium chloride added to drinking water for the duration of the study. For sham UCAO (sUCAO) surgery, a ventral midline incision was made to expose the right carotid artery. After isolation of the right carotid artery sham-operated rats were fed a regular rodent diet (RD/sUCAO) with tap water for the duration of the study. All animals in the study survived to the sixteenth week of life at which point they were euthanized. In our previous studies with these animals, high morbidity and mortality ensued after the midpoint of the sixteenth week for the SHR/SP JPD/UCAO groups.8

Magnetic Resonance Imaging Experiments

Rats with LiPc implants were placed in a custom-built holder and moved to the isocenter of the magnet before imaging. Throughout the imaging session, animals were anesthetized with isoflurane (4% in N2O:O2 (70:30%) for induction and 2% in N2O:O2 (70:30%) for maintenance), and the heart and respiratory rates were monitored in real time. Magnetic resonance imaging was performed on a 4.7T MRI dedicated MR scanner (Bruker Biospin, Billerica, MA, USA), equipped with a 500-mT/m (rise time 80 to 120 ms) gradient set for performing small animal imaging, and a small-bore linear RF coil (ID 72 mm). Implant position was confirmed using T2-weighted 2D RARE (rapid acquisition with relaxation enhancement) imaging using the following parameters: repetition time/echo time, 4,000/65 ms; field of view, 2.5 cm × 2.5 cm; slice thickness, 1.0 mm; slice gap, 0.5 mm; number of slices, 10; matrix, 256 × 128; number of averages, 20; receiver bandwidth, 50 kHz.

Measurement of Cerebral White Matter pO2 by Electron Paramagnetic Resonance Oximetry with Lithium Phthalocyanine

For in vivo measurement of local cerebral WM pO2 in the anesthetized rat, EPR oximetry was conducted according to previously described methods21, 22, 23 with some modification. Throughout the weekly EPR measurements, animals were anesthetized with isoflurane (4% in N2O:O2 (70:30%) for induction and 2% in N2O:O2 (70:30%) for maintenance), and maintenance of core (rectal) temperature at 37.5±0.5°C was performed using a heat lamp. Briefly, an external loop resonator was placed over the position where LiPc was implanted, and an EPR spectrum was recorded using a Bruker EleXsys E540 EPR spectrometer equipped with an L-band bridge (Bruker Instruments, Billerica, MA, USA). The resonator has advanced automatching and autotuning capabilities that correct for any slight animal movements. The EPR spectrum was acquired with a scan time of 40 seconds, and 5 scans were obtained and averaged to produce a significant signal-to-noise ratio to allow accurate fitting. The peak-to-peak linewidth of the spectrum was obtained via computer linefitting, and converted to pO2 values according to a calibration curve for the oximetry probe LiPc as previously described.21, 22, 23 Electron paramagnetic resonance acquisition parameters: microwave power of 18 mW, a microwave frequency of 1.07 GHz, a center magnetic field strength of 380 G, a scan range of 1.0 G, and a modulation amplitude of less than one-third of the intrinsic EPR linewidth. Interstitial pO2 was measured continuously throughout the study and reported at the time points shown.

Histologic Staining

Paraffin-embedded brain sections were assessed for WM damage and blood vessel changes by hematoxylin and eosin (H&E) staining using standard techniques employing Lillie's variant of Mayer's hemalum (Lillie-Mayer) and eosin/phloxine. Briefly, tissues were rehydrated through alcohol series and exposed to hematoxylin for nuclear staining. Slides were rinsed in tap water and differentiated using acid alcohol and Scott's tap water substitute. Eosin/phloxine counterstaining was performed followed by serial alcohol dehydration, clearing with xylenes, and coverslipping with DPX. Stained slides were viewed and imaged an Olympus BX-51 (Center Valley, PA, USA) bright field microscope equipped with an Optronics digital camera.

Statistics

One-way and two-way non-parametric ANOVAs for comparison of weekly measurements between the various groups were used with Tukey's (two-way) and Dunn's (one-way) corrections for multiple t-tests. Values are expressed as mean±standard error of the mean. Significance was considered with P<0.05.

Results

Long-Term Stability and Sensitivity of lithium phthalocyanine Crystal to Cerebral Tissue pO2 in the White Matter of Wistar-Kyoto Rats

Initially, we established the stability of the LiPc crystal in the WM by measuring tissue pO2 in 12-week-old WKY rats after implantation of the LiPc crystal in the WM of the brain. The precise location of the crystal in the WM was visualized in coronal sections obtained after death (Figure 1A) and with MRI during life (Figure 1B).

Figure 1.

Figure 1

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Magnetic resonance (MR) image and brain section of rat brain implanted with lithium phthalocyanine (LiPc) crystal. (A) Coronal brain section showing the location of the LiPc crystal in the white matter (WM). (B) 4.7T magnetic resonance imaging (MRI) (T2-weighted 2D RARE) indicating the location in the WM of the LiPc crystal in an anesthetized rat. Arrows indicate LiPc in the WM. RARE, rapid acquisition with relaxation enhancement.

We made repeated measurements of cerebral pO2 in the WM over a 4-week period from 12 to 16 weeks of age (Figure 2A). Cerebral pO2 remained constant over the 4 weeks when breathing 30% oxygen under anesthesia (2% isoflurane in N2O:O2 70:30%). These results agree with previous reports showing stability of the crystal and feasibility of repeated measurements up to 30 days in the cortex,26 but this was the first demonstration of stable measurements in WM over several weeks.

Figure 2.

Figure 2

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The change in interstitial pO2 levels in the white matter of Wistar-Kyoto (WKY) rats at different weeks of age postimplantation and the relationship of the cerebral pO2 of rats at 16 weeks of age with oxygen content in breathing gas. Lithium phthalocyanine crystal was implanted in the rat brain 48 hours before the first electron paramagnetic resonance (EPR) measurement. EPR spectra were collected from anesthetized rats breathing 30% oxygen and converted to pO2 (mm Hg). (A) Interstitial pO2 levels collected at weeks 12, 15, and 16 of age from anesthetized rats. (B) Sequentially, interstitial pO2 levels as anesthetized rats inhaled gas mixtures of 20%, 30%, and 50% O2 and converted to pO2 (mm Hg). There was a 15-minute wait after each change in O2 to allow pO2 to reach equilibrium. Asterisks indicate significant (P<0.05) difference when compared with other respired O2%. Data are expressed as the mean±SEM (N=5).

To assess the sensitivity of LiPc in the WM to O2 in a long-term study, the linewidth response of implanted LiPc in WKY rats to respired O2 was measured as the breathing gas was increased incrementally from 20% to 50% O2 at age 16 weeks, 4 weeks after implantation. The corresponding changes in linewidth were obtained and subsequent measured WM tissue pO2 values were calculated. Figure 2B shows that WM tissue pO2 increased with increasing level of O2 in the respiration gas, and the values obtained are consistent with previous findings in rats.21, 22, 23 These results suggest that EPR oximetry with LiPc can accurately and sensitively measure WM tissue pO2 over extended period of time.

Cerebral pO2 in the White Matter of Hypertensive Stroke-Prone Rats

After determination that EPR oximetry with LiPc accurately measures tissue O2 in the WM of WKY rats, we measured pO2 in the WM of SHR/SP over the long term. Electron paramagnetic resonance oximetry measurements obtained at 6 to 8 weeks were consistent with pO2 values obtained from WKY rats and the literature.21, 22, 23 At 9 weeks of age, a gradual increase in WM pO2 was observed that continued to rise weekly up to 12 weeks of age. At age week 12, a significant 32% increase in pO2 in the WM of SHR/SP rats was observed compared with baseline measurements at age weeks 6 to 8 as shown in Figure 3A.

Figure 3.

Figure 3

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The change in interstitial pO2 levels in the white matter (WM) of spontaneously hypertensive stroke-prone rat (SHR/SP) rats postimplantation from 6 to 12 weeks of age and after unilateral carotid artery occlusion (UCAO) surgery with Japanese permissive diet (JPD) (JPD/UCAO) from 12 to 16 weeks of age. Lithium phthalocyanine crystal was implanted in the rat brain 48 hours before the first electron paramagnetic resonance (EPR) measurement. EPR spectra were collected from anesthetized rats breathing 30% oxygen and converted to pO2 (mm Hg). (A) Interstitial pO2 levels in WM of SHR/SP without surgery or diet from 6 to 12 weeks of age. Data are expressed as the mean±SEM (N=8). Asterisk (*) indicates a significant (P<0.05) difference when compared with weeks 6, 7, and 8. (B) Interstitial pO2 levels in the WM of JPD/UCAO rats after UCAO surgery with JPD from 12 to 16 weeks of age. Data are expressed as the mean±SEM (N=8). Asterisk (*) indicates a significant (P<0.05) difference when compared with weeks 13 to 16.

At 12 weeks of age one group of SHR-SP rats was given the JPD with UCAO surgery (JPD/UCAO). For comparison, two control groups were used: (1) sUCAO surgery involving a neck incision with manipulation of the carotid along with a regular rodent diet (RD/sUCAO) and (2) SHR/SP of the same age but without any intervention (no intervention).

Among the three groups of SHR/SP with different interventions, the maximum decrease in pO2 occurred in the JPD/UCAO rats (Figure 3B). In the JPD/UCAO group the pO2 at 12 weeks was significantly different from all other weeks, which was similar to the RD/sUCAO group. In the no intervention group, there was a significant difference only between weeks 12 and 13. Sham surgery animals showed an intermediate decrease in pO2, which was significantly different from the no intervention SHR/SP in the fifteenth and sixteenth weeks; there were significant differences seen in the JPD/UCAO compared with untreated controls after the thirteenth week (Table 1).

Table 1. The change in interstitial pO2 values, obtained with EPR and LiPc in the white matter, for the three groups of SHR-SP rats used in the study: (1) JPD/UCAO; (2) RD/sUCAO; and (3) No intervention.

Group 12 weeks 13 weeks 14 weeks 15 weeks 16 weeks
JPD/UCAO (N=17) 37.1±1.2 19.5±1.2 15.1±1.2 14.9±1.2 13.3±1.2
RD/sUCAO (N=8) 41.8±2.5 19.7±3.4 18.0±3.4 16.8±3.7a 15.4±3.8a
No treatment (N=6) 34.1±2.7 24.2±1.2 26.0±1.2b 25.8±1.2b 25.6±1.2b
P (two-way ANOVA) n.s. n.s. 0.005 0.006 0.003
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EPR, electron paramagnetic resonance; LiPc, lithium phthalocyanine; SHR-SP, spontaneously hypertensive stroke-prone rat; UCAO, unilateral carotid artery occlusion; JPD, Japanese permissive diet; sUCAO, sham UCAO; RD, regular rodent diet; ANOVA, analysis of variance.

a

Significant difference between sham surgery and no intervention animal groups.

b

Significant difference between JPD/UCAO and no intervention animal groups.

Histopathology Assessment of Tissue and Vascular Damage

To assess whether SHR/SP with or without JPD/UCAO treatment would spontaneously develop neuropathologic lesions and vascular morphologic changes in WM, and their correlation with the changes in WM pO2 shown by EPR analysis, H&E staining of the brain sections of the SHR/SP animals was conducted, and WKY rats at age 12 weeks were used as a normal animal control. The rats, which were used for histopathologic assessment of tissue and vascular damage, were not subjected to EPR measurement. Blinded examination of H&E-stained histologic sections by a registered neuropathologist and a neurologist revealed that no significant morphologic difference was observed in corpus callosum or internal/external capsules (data not shown) in untreated SHR/SP up to age 16 weeks compared with WKY. By 16 weeks, the naïve SHR/SP rats show no evidence of BBB damage and hemorrhages in WM (Figure 4). In contrast, WM damage, BBB disruption, hemorrhages, and inflammatory cells were seen in age-matched SHR/SP 4 weeks after JPD/UCAO (Figure 5), which was accompanied with the drastic reduction in tissue pO2 in the white matter at this time point (Figure 3B).

Figure 4.

Figure 4

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Histologic examination of white matter in naïve Wistar-Kyoto (WKY) rats and spontaneously hypertensive stroke-prone rat (SHR/SP) rats. Left top panel: Hematoxylin and eosin (H&E)-stained brain section from a representative WKY rat, aged 12 weeks. Right top panel: H&E-stained brain section from a representative SHR/SP rat, aged 7 weeks. Bottom panels: H&E-stained brain sections from representative SHR/SP rats, aged 12 and 16 weeks, respectively. Insert shows vessels (arrows) in corpus callosum. CTX, cortex; CC, corpus callosum; Hip, hippocampus. Scale bar=100 μm (for all panels).

Figure 5.

Figure 5

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Blood–brain barrier damage 4 weeks after JPD/UCAO in spontaneously hypertensive stroke-prone rat (SHR/SP) rats. Hematoxylin and eosin (H&E)-stained sections revealed hemorrhages surrounding vessels in the area of nonoccluded (left) sides of corpus callosum (LCC) in JPD/UCAO group. Perivascular infiltrate of inflammatory-like cells (inserts 1 and 2) in both occluded (RCC) and nonoccluded hemispheres. Scale bars=100 μm (left panels) and 50 μm (right panels). UCAO, unilateral carotid artery occlusion; JPD, Japanese permissive diet.

Discussion

This is the first report to use EPR oximetry, a versatile method for highly sensitive and repetitive measurements of pO2 in the brain, to investigate tissue pO2 in the WM, and specifically, of normotensive WKY and hypertensive SHR/SP rats. After implantation of the LiPc crystal in the WM, stable measurements were made over 6 weeks in the chronically implanted animals. Increasing the ambient O2 produced an increase in the tissue pO2. After establishing the stability and responsiveness of the implanted crystals, and verifying the location with MRI and/or autopsy, we measured pO2 in the SHR/SP rats that were fed a regular diet between 6 and 12 weeks of age; showing that the pO2 up to 8 weeks was similar to age-matched WKY. However, at 12 weeks tissue pO2 was significantly increased in WM of SHR/SP. By week 13 the WM pO2 in JPD/UCAO rats decreased to hypoxic levels, and remained low until 16 weeks when they died. Untreated SHR/SP showed a similar increase in pO2 at 12 weeks, but had less of a decrease at 13 weeks and remained alive. The continued decrease in pO2 in the JPD/UCAO group was associated with vascular damage and death by 16 weeks, which contrasted with the normal histology in the untreated rats in spite of the decrease in pO2.

White matter is vulnerable to hypoxic-ischemic injury due to the watershed nature of the cerebral blood flow to the deep structures in the brain, making it a site of injury when hypoxia occurs.27 Untreated SHR/SP generally develops hemorrhages after 1 year of age, which may be accompanied by changes in the WM. Hypertension rises during the first 12 weeks of life, plateauing after that age. However, when SHR/SP is fed a high salt, low protein diet along with UCAO, there is demyelination and vascular damage that leads to death after 16 weeks of age. In the JPD/UCAO animals, damage to the WM appears between weeks 12 and 16. White-matter pathology resembles that seen in VCI patients with small vessel disease, making the SHR/SP a relevant model for studying the human disease found in the elderly patients with hypertension-related WMHs.8 Earlier, we showed that MMP-9 mediated neuroinflammation in SHR/SP/JPD/UCAO was associated with BBB opening, demyelination, and oligodendrocyte death. Using EPR to directly measure pO2, we showed for the first time the presence of hypoxia in the deep WM. Although ischemic lesions and damage have been observed using MRI techniques, direct measurement of tissue pO2 in the WM has not been explored and the effects that ischemia exerts on WM have been seldom studied.28, 29, 30 Using the unique capability of EPR oximetry combined with LiPc to measure localized interstitial pO2 in living animals, we were able to assess cerebral pO2 in the WM of rats in vivo, for the first time, over a longitudinal study.

The measurement of pO2 is fundamental in such disciplines as ischemia, altitude adaptation, tumor growth, and angiogenesis and the ability to make multiple measurements in small brain regions is a unique advantage of EPR oximetry.20, 21, 22, 23 Confirmation of implant position validated that pO2 measurements were specific to the site of interest. Brain tissue pO2 measurements can be ascertained in the same animal and at the same location in the brain WM. We showed that pO2 measurements were stable over 4 weeks in WKY, and that the LiPc crystal was responsive to increases in ambient O2. Values obtained in the WM of WKY rats over this time course and in response to respired O2 were consistent with previous literature measurements obtained in the gray matter of Sprague-Dawley rats under anesthesia and while breathing various O2 concentrations.23, 31 Of note, although this paper focuses on longitudinal WM changes, it will be of interest to determine differences, if any, between WM compared with gray-matter pO2 in the SHR/SP model using EPR oximetry in future endeavors.

We measured pO2 in the WM of SHR/SP at 6 weeks of age before the onset of severe hypertension; all of the animals in the three groups showed similar levels of pO2 up to 12 weeks. The observed cerebral pO2 for SHR/SP at 6 to 8 weeks of age was comparable to measurements of WKY. However, we observed an unexpected increase in WM pO2 from weeks 9 to 12 in SHR/SP, which was followed by a dramatic decrease in cerebral WM pO2 in SHR/SP with JPD/UCAO between 12 and 16 weeks of age. All three groups of SHR/SP had a decline in pO2 beginning at week 13, which was greatest in those in the JPD/UCAO group and least in the untreated group. Hypertension narrows the lumen of the vessels supplying blood to the brain, increasing the resistance to flow, which could explain the limited O2 delivery to the brain.32

Although SHR/SP rats develop hypertension between 8 and 12 weeks of age, the increased incidence of spontaneous stroke (95% die of stroke) in untreated animals occurs much later, at ∼1 year of age; by studying SHR/SP at 12 weeks and initiating JPD and UCAO at that time, we avoided the high incidence of stroke. In addition, others have shown a reduction in cerebral blood flow in conjunction with hypertension in SHR/SP with a marked decrease after 12 weeks, which is consistent with the timing we observed in hypoxic changes.33, 34, 35

In conclusion, we used the SHR/SP with poor diet and carotid occlusion as a model for WM disease in VCI patients, and showed for the first time using direct pO2 measurements with EPR a significant reduction in the O2 in the deep WM. Our results suggest that changes in WM pO2 may contribute to the selective injury in the deep WM unrelated to frank ischemic infarcts. This could explain the vulnerability of patients with limited reserve capacity that may have chronic or intermittent hypoxia secondary to the hypertensive vascular disease. Electron paramagnetic resonance allowed for multiple measurements over several months, showing that compared with normotensive WKY, SHR/SP experiences broad changes in tissue O2 in the brain and specifically in the WM over the course of 16 weeks of age. We also present novel findings that the combination of EPR oximetry with the probe LiPc can make direct, accurate, and repeated measurements of WM brain tissue pO2 in small animals. This innovative technique could become an important tool to address the fundamental questions regarding the role of oxygenation in the WM of the brain.

The authors declare no conflict of interest.

Footnotes

This work was partially supported by grants from the NIH (RO1NS045847 to GR and P30GM103400 to KJL) and a pilot project grant from an NIH CTSA at UNM (UL1TR000041).

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