(Stroke. 1995;26:1444-1452.)
© 1995 American Heart Association, Inc.
Articles |
From the Thoralf M. Sundt Jr Neurosurgical Research Laboratory, Mayo Clinic and Mayo Graduate School of Medicine, Rochester, Minn.
| Abstract |
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Methods Twenty fasted rabbits under 1.0% halothane anesthesia were divided into four groups: (1) nonischemic controls, (2) 60 minutes of uninterrupted focal ischemia, (3) 2x30-minute periods of focal ischemia separated by a 5-minute reperfusion, and (4) 4x15-minute periods of focal ischemia separated by three 5-minute reperfusion periods. Focal ischemia was produced by occlusion of both the middle cerebral and ipsilateral anterior cerebral arteries. After the final occlusion, there was a 3-hour reperfusion period in all groups. Regional cerebral and cortical blood flow, brain pHi, and NADH fluorescence were measured with in vivo panoramic fluorescence imaging.
Results During occlusion, regional cerebral and cortical blood flows and NADH fluorescence values were not different among the groups. Brain pHi was significantly lower in the 4x15-minute group compared with the 1x60-minute group (6.57±0.02 versus 6.73±0.06; P<.03) but not significant when compared with the 2x30-minute group. During the short reperfusion periods, all parameters returned to normal except for NADH fluorescence levels, which remained elevated. During the postischemic final reperfusion period, there was a mild brain alkalosis of approximately 7.1 in all groups. There were no significant differences in NADH fluorescence among groups during the final reperfusion. Regional cerebral and cortical blood flow returned to near normal values in all groups.
Conclusions This study demonstrates that intermittent reperfusion during temporary focal ischemia has different effects on the intracytoplasmic and the intramitochondrial compartments: worsening of brain cytoplasmic pHi but no significant differences in the oxidation/reduction level of mitochondrial NADH.
Key Words: brain intracellular pH cerebral ischemia, focal NADH fluorescence reperfusion
| Introduction |
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Alternatively, at least one study suggests that brief periods of intermittent reperfusion during temporary focal ischemia designed to mimic the typical operative scenario decrease infarction size.15 The possible beneficial effects of intermittent reperfusion may include restoration of necessary metabolic substrates, waste product removal, and the mitigation of the development of an environment conducive to the production of free radicals.7 16 17 The purpose of this experiment was to examine the serial changes in pHi, regional cerebral (133Xe) or cortical (umbelliferone) blood flow, and the NADH oxidative/reduction state during intermittent reperfusion comparable with the operative setting. The hypothesis tested in this study was that brief intermittent reperfusion results in improved normal metabolic function as assessed by brain pHi and NADH fluorescence.
| Materials and Methods |
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Catheters were inserted into the right femoral artery and vein for monitoring blood pressure and sampling arterial blood gases and for administration of drugs. A PE-50 catheter was inserted into the right lingual artery so that its tip was located at the origin of the external carotid artery for the retrograde delivery of the indicator umbelliferone and 133Xe into the internal carotid artery.
The skin, subcutaneous tissue, and muscle were excised over the right supraorbital ridge and parietal area. A craniectomy was performed using a high-speed air drill (Hall Surgical, Division of Zimmer) with the aid of an Olympus operating microscope. The majority of the frontal and parietal cortex was exposed for imaging. The dura was removed and carefully cauterized at the margins of the craniectomy, then covered with plastic wrap to prevent surface oxygenation and to keep the brain moist. Blood loss for the surgical preparation did not exceed 5 mL.
After the surgical preparation, the animals were moved from the operating table and placed on an intravital-type microscope stand. The microscope was focused on an area centered about the suprasylvian gyrus with 1.5 cm2 of cortex imaged for pHi, rCtBF-UM, and NAD/NADH measurements. Arterial blood pressure was measured by a Statham strain gauge attached to the femoral artery catheter and recorded on a Grass model 78 polygraph. The animals were kept normothermic by the use of a heating blanket (K-Pad, Gorman-Rupp), and body temperature was monitored with a rectal digital thermometer. Arterial PaCO2, PaO2, and pH measurements were performed on a London Radiometer blood gas analyzer (PHM-73).
After control measurements were performed, focal cerebral ischemia was produced by occlusion of the proximal middle and anterior cerebral arteries using Mayfield miniature aneurysm clips. Twenty rabbits were divided into four groups of five each: (1) a nonischemic control observed for 4 hours; (2) a control of 60 minutes of straight ischemia followed by 3 hours of reperfusion; (3) two 30-minute periods of ischemia separated by 5 minutes of reperfusion followed by 3 hours of reperfusion; and (4) four 15-minute periods of ischemia each separated by 5 minutes of reperfusion followed by a final 3-hour period of reperfusion. All animals underwent a high PaCO2 reactivity test to ensure normal function before occlusion. A PaCO2 reactivity test was also performed at the end of 3 hours of reperfusion.
In Vivo Video Fluorescence Instrumentation
Instrumentation was designed to perform serial panoramic video
imaging of cortical brain pHi and regional cortical blood
flow with umbelliferone fluorescence.18 The
optical characteristics were such that the majority of the entire
hemisphere could be studied simultaneously through a large
craniectomy. The use of umbelliferone as a noninvasive in vivo
technique for measuring brain pHi and cortical blood flow
has been previously described.18 19 20 21 Umbelliferone is
nontoxic, fat-soluble, and freely diffuses across the blood-brain
barrier, and it rapidly equilibrates across cell membranes and is
distributed through the cytoplasm as an uncharged
molecule.18 Umbelliferone was prepared for injection by
dissolving 0.2 g of indicator in 200 mL 5% glucose-saline solution at
90°C for 30 minutes. The solution was then filtered through a
0.22-µm mesh filter before injection. The volume of injectate was 1.5
mL in this study.
The pH-sensitive indicator umbelliferone has two fluorophors, anionic and isobestic. The anionic and isobestic forms are excited at 370 nm and 340 nm, respectively, and have a common emission at 450 nm. The fluorescence of the anion varies directly with pH, whereas the fluorescence of the isobestic form varies directly only with the indicator concentration. Therefore, it is possible to create a nomogram from the ratio of 340-nm to 370-nm excitations to determine brain pHi. Acquired images were corrected for background NADH fluorescence before processing. NADH fluorescence images were stored for later analysis of mitochondrial function. The images from the 340-nm excitation were processed to compute rCtBF-UM using the 1-minute initial slope index. The partition coefficient for umbelliferone is one.21 The rCtBF-UM image was then displayed and stored on tape for final analysis. For processing of the pHi image, ratios of the paired images from the 340-nm and 370-nm excitations were made, and the resultant pHi image was then displayed and stored on tape for final analysis.
Umbelliferone has been shown to be a reliable indicator of regional cortical blood flow. Anderson et al21 have made a comparative analysis of the different techniques of measuring rCBF compared with that of umbelliferone. They found that a distinction can be made between rCBF as measured by radiolabeled compounds and that of regional cortical blood flow as measured by umbelliferone. rCBF by definition is defined as areas of flow that contain major vessels as well as capillaries and arterioles as measured by radiolabeled compounds. Regional cortical blood flow as measured by umbelliferone is defined as those areas that are relatively avascular and contain only capillaries. The imaging system allows the measurement of regional cortical blood flow by setting the number of pixels to cover specified areas.
133Xe Regional Cerebral Blood Flow
Measurements
133Xe rCBF was measured using a CdTe (Cadmium
Telluride) detector (RMD) system. The detector has a measurement volume
of 0.50 mm3. The window discriminator was set at 76 keV and
200 keV to minimize Compton scatter.22 The resultant
counts were recorded on a strip-chart recorder. The 1-minute
initial slope index was used to calculate rCBF.23 The
partition coefficient (
) used for 133Xe was
0.63.24
Statistical Analysis
Because of anatomic variation of the microvasculature from
animal to animal, single points along an x-y
coordinate cannot be averaged frame by frame from different animals at
the same time period. Therefore, measurements of regional
pHi, regional cortical blood flowumbelliferone
(rCtBF-UM), and NADH fluorescence were made in areas devoid of
major vessels. Measurements were made over these relatively avascular
areas by averaging 10 000 to 15 000 pixels (22 500 to 37 500
µm2), and the mean and standard error were tabulated.
Student's t test was used for comparison between the
nonischemic control group and each of the ischemic
groups with correction for multiple comparisons. ANOVA followed by
Tukey's test for multiple comparison was used to test the statistical
significance of differences among the four groups studied. A value of
P<.05 was considered significant.
| Results |
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Group 1: 60-Minute Straight Occlusion Followed by 3-Hour
Reperfusion
Intracellular Brain pH
Baseline brain pHi was uniform over the entire exposed
cortex measuring 7.02±0.02. After 15 minutes of focal cerebral
ischemia, pHi fell significantly
(P<.005) to 6.69±0.02 and remained at this level for the
next 45 minutes. Thirty minutes after restoration of flow, brain
pHi showed a rise toward normalization with an initial
value of 7.06±0.03, not significantly different from the baseline
control values, and remained close to this level for the next 2.5 hours
(Fig 1A
).
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Regional Cortical Blood FlowUmbelliferone
Baseline rCtBF-UM measured 48.1±3.9 mL · 100
g-1 · min-1. Fifteen minutes after
occlusion, rCtBF-UM fell significantly (P<.005) to
18.0±2.3 mL · 100 g-1 · min-1 and
remained at this level for the next 45 minutes. Thirty minutes after
the restoration of blood flow, rCtBF-UM rose to 37.9±3.9 mL · 100
g-1 · min-1 and was relatively constant
over the 2.5 hours, not significantly different from baseline control
values (Fig 1B
).
Regional Cerebral Blood Flow133Xe
Baseline rCBF-133Xe measured 57.6±0.6 mL · 100
g-1 · min-1. Fifteen minutes after
occlusion, rCBF-133Xe fell significantly
(P<.05) to 32.9±2.7 mL · 100
g-1 · min-1 and remained at this level
for the next 45 minutes. Thirty minutes after the restoration of blood
flow, rCBF-133Xe rose to 47.6±5.0 mL · 100
g-1 · min-1 and was relatively constant
over the 2.5 hours, not significantly different from baseline control
values (Fig 1D
).
NADH Fluorescence
The baseline NADH fluorescence gray scale level was
41.7±0.4. Fifteen minutes after occlusion, NADH fluorescence
increased significantly by 136±11.0% (P<.05) when
compared with baseline control values and remained at this level for
the next 45 minutes. Thirty minutes after the restoration of blood
flow, NADH fluorescence decreased to 126.0±3.0% of baseline
control. After this time period, NADH fluorescence declined
slightly for the remainder of the experiment but remained significantly
greater than baseline control values (P<.005) (Fig 1C
).
Group 2: Two 30-Minute Occlusions Followed by 3-Hour
Reperfusion
Intracellular Brain pH
Baseline brain pHi was uniform over the entire exposed
cortex measuring 7.06±0.02. After 30 minutes of focal
ischemia, pHi fell significantly
(P<.005) to 6.78±0.07. After 5 minutes of reperfusion,
brain pHi was 7.00±0.03, not different from baseline
values. Brain pHi at 30 minutes from the second
ischemic insult was 6.73±0.03 (P<.005). Thirty
minutes after restoration of blood flow, brain pHi rose to
7.05±0.04 and was slightly alkalotic for the next 2.5 hours, but it
was not significantly different from baseline control values (Fig 2A
).
|
Regional Cortical Blood FlowUmbelliferone
Baseline rCtBF-UM measured 45.8±2.6 mL · 100
g-1 · min-1. Thirty minutes after
occlusion, rCtBF-UM fell significantly (P<.005) to
16.9±1.7 mL · 100 g-1 · min-1. After
5 minutes of reperfusion, rCtBF-UM rose to 36.8±2.3 mL · 100
g-1 · min-1, not different from
baseline control values. Thirty minutes from the second
ischemic insult, rCtBF-UM fell to 18.7±4.3
(P<.005). Thirty minutes after the restoration of blood
flow, rCtBF-UM rose to 24.0±1.11 mL · 100
g-1 · min-1, significantly
different from baseline control values (P<.05). It
continued to rise and at 1 hour after restoration of flow, it became
not significantly different from baseline control values (Fig 2B
).
Regional Cerebral Blood Flow133Xe
Baseline rCBF-133Xe measured 53.4±1.9
mL · 100 g-1 · min-1. Thirty minutes
after occlusion, rCBF-133Xe fell significantly
(P<.05) to 33.4±4.7 mL · 100
g-1 · min-1. After 5 minutes of
reperfusion, rCBF-133Xe rose to 44.3±3.3 mL · 100
g-1 · min-1, not different from
baseline control values. Thirty minutes from the second
ischemic insult, rCBF-133Xe fell to 34.8±3.3
(P<.05). Thirty minutes after the restoration of blood
flow, rCBF-133Xe rose to 38.5±2.2 mL · 100
g-1 · min-1, not different from
baseline control values. It remained at this level for the duration of
the experiment (Fig 2D
).
NADH Fluorescence
Baseline NADH fluorescence gray scale level was
41.3±0.5. Thirty minutes after occlusion, NADH fluorescence
increased significantly to 132.0±5.0% (P<.005) when
compared with baseline control values. After 5 minutes of reperfusion,
NADH fluorescence decreased to 124.0±4.0% of baseline
control, still significantly different (P<.005). Thirty
minutes from the second ischemic insult, NADH
fluorescence rose to 135.0±7.0% of baseline controls
(P<.005). Thirty minutes after the restoration of blood
flow, NADH fluorescence decreased to 129.0±5.0% of baseline
control but was still significantly different (P<.005).
After this time period, NADH fluorescence declined for the
remainder of the experiment and became not significantly different 1.5
hours after restoration of flow (Fig 2C
).
Group 3: Four 15-Minute Occlusions Followed by 3-Hour
Reperfusion
Intracellular Brain pH
Baseline brain pHi was uniform over the exposed
cortex measuring 7.08±0.01. After 15 minutes of focal
ischemia, pHi fell significantly
(P<.005) to 6.65±0.03. After 5 minutes of reperfusion,
brain pHi was 6.94±0.03, not different from baseline
values. After three 15-minute ischemic insults and three
5-minute reperfusions, brain pHi at 15 minutes from the
fourth ischemic insult was 6.57±0.02 (P<.005).
Thirty minutes after restoration of blood flow, brain pHi
rose to 7.10±0.02 and became significantly alkalotic
(P<.05) for the next 2.5 hours (Fig 3A
).
|
Regional Cortical Blood FlowUmbelliferone
Baseline rCtBF-UM measured 43.5±3.3 mL · 100
g-1 · min-1. After 15 minutes of focal
ischemia, rCtBF-UM fell significantly (P<.005) to
20.7±3.1 mL · 100 g-1 · min-1. After
5 minutes of reperfusion, rCtBF-UM rose to 48.1±9.2 mL · 100
g-1 · min-1, not different from
control baseline values. After three 15-minute ischemic insults
and three 5-minute reperfusions, rCtBF-UM at 15 minutes from the fourth
ischemic insult was 21.2±2.2 mL · 100
g-1 · min-1 (P<.005). Thirty
minutes after restoration of blood flow, rCtBF-UM rose to 36.2±4.83
mL · 100 g-1 · min-1, not
different from baseline control values. It remained at this level for
the duration of the experiment (Fig 3B
).
Regional Cerebral Blood Flow133Xe
Baseline rCBF-133Xe measured 49.1±1.9 mL · 100
g-1 · min-1. After 15 minutes of focal
ischemia, rCBF-133Xe fell significantly
(P<.005) to 27.2±1.4 mL · 100
g-1 · min-1. After 5 minutes of
reperfusion, rCBF-133Xe rose to 44.8±3.4 mL · 100
g-1 · min-1 but was not different from
baseline control values. After three 15-minute ischemic insults
and three 5-minute reperfusions, rCBF-133Xe at 15 minutes
from the fourth ischemic insult was 27.2±1.1 mL · 100
g-1 · min-1 (P<.005). Thirty
minutes after restoration of blood flow, rCBF-133Xe rose to
38.1±3.7 mL · 100
g-1 · min-1, not different from
baseline control values. It remained at this level for the duration of
the experiment (Fig 3D
).
NADH Fluorescence
The baseline NADH fluorescence gray scale level was
42.4±0.9. After 15 minutes of focal ischemia, NADH
fluorescence increased significantly (P<.005) to
127.0±4.0% of baseline control values. After 5 minutes of
reperfusion, NADH fluorescence decreased to 122.0±4.0% of
baseline control, significantly different from preocclusion values
(P<.05). After three 15-minute ischemic insults and
three 5-minute reperfusions, 15 minutes from the fourth
ischemic insult NADH fluorescence increased to
127.0±6.0% of baseline control values (P<.05). Thirty
minutes after restoration of blood flow, NADH fluorescence
decreased to 124.0±7.0% of baseline control, still significantly
different (P<.05) from preocclusion. After this time
period, NADH fluorescence declined for the remainder of the
experiment and became not significantly different 1 hour after
restoration of flow (Fig 3C
). An intergroup comparison of NADH
fluorescence during the final reperfusion period was made among
all three groups. This intergroup analysis demonstrated no
significant difference in the NAD/NADH redox state during the final
postischemic reperfusion period.
PaCO2 Reactivity
The PaCO2 reactivity test performed at
the beginning and at the end of the experiment demonstrated little or
no change in pHi in all groups studied. The reactivity with
rCtBF-UM showed no significant difference in the 60-minute
straight-occlusion group. In the 2x30-minute reperfusion group, there
was a 6% decline in 133Xe-rCBF reactivity, which was not
significantly different. In the 4x15-minute reperfusion group, there
was a 33.5% decline in 133Xe-rCBF reactivity
(P<.01) compared with baseline preischemic
values. However, an intergroup analysis of
postischemic CO2 reactivity showed no
significant differences.
| Discussion |
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The regulation of brain pHi is primarily dependent on the Na+/H+ pump located along the cytoplasmic membrane and intracellular organic bases.25 26 27 Conceptually, the measurement of pHi can be considered a cytoplasmic marker of cellular function. The presence of a severe brain acidosis and the accumulation of lactic acid may determine in part the differences between selective neuronal injury and pannecrosis.28 29 Intracellular acidosis has the following postulated detrimental effects: (1) increasing glial edema with subsequent extravascular capillary-bed compression leading to reductions in potential collateral blood flow,27 30 31 (2) the inhibition of mitochondrial respiration,32 33 (3) the retardation of NADH regeneration,34 and (4) the promotion of free radical generation by facilitating the Haber-Weiss reaction.35 36 It should also be recognized, however, that there is some experimental evidence that suggests that acidosis might be neuroprotective by blocking the N-methyl-D-aspartategated calcium channel.37 38 These controversial data regarding the effects of brain acidosis during ischemia are reflected in the inconclusive results on the effects of hyperglycemia during focal ischemia. It has long been thought that hyperglycemia increases brain acidosis and injury through increased lactic acid production.39 40 However, the effects of hyperglycemia during focal cerebral ischemia are inconclusive with both an increase in, a decrease in, and no effect on infarction sizes being reported with hyperglycemia.41 42 43 Therefore, the significance of the observation that brief periods of intermittent reperfusion in this present experiment led to a worsening in acidosis during the ischemic period remains indeterminate.
The redox state of NAD/NADH is an assessment of mitochondrial function.44 45 The intergroup comparison in this experiment demonstrated no significant differences in NADH fluorescence during the final reperfusion. This would support the findings of Selman et al,46 who measured ATP levels in both Wistar and spontaneously hypertensive rats subjected to intermittent or single middle cerebral artery occlusion. Their metabolic data in the spontaneously hypertensive rats demonstrated no significant differences in ATP levels between the single and multiple occlusion groups. This would suggest that intermittent reperfusion did not prevent critical ATP depletion. It should be noted that, in their study, the intermittent reperfusion group had relatively preserved ATP levels in the ischemic core region, which would agree with the NAD/NADH data provided in the present experiment. Surprisingly, in their study this preservation of ATP did not attenuate the degree of histological injury.
A review of the current literature examining the effects of intermittent reperfusion in focal cerebral ischemia remains indeterminate in providing a definitive answer regarding the potential benefits or adverse effects of brief periods of reperfusion. For example, Sakaki et al47 studied the effects of three 20-minute episodes of middle cerebral artery occlusion compared with a straight 1-hour period of ischemia in cats. They noted that intermittent occlusion led to a reduction in the degree of pathological injury. Unfortunately, the time of the reperfusion periods was not specified. Steinberg and colleagues48 provided evidence that intermittent reperfusion led to a reduction in the degree of cortical neuronal injury in a rabbit model of focal cerebral ischemia. However, there was no difference in the degree of striatal ischemic injury. In a prior study from this laboratory,15 10-minute periods of ischemia separated by 5 minutes of reperfusion led to a significant reduction in infarct size when compared with single occlusion times of 60, 90, or 120 minutes. As indicated above, the more recent study by Selman and colleagues46 found no difference in histopathology and ATP, lactate, and glucose levels between single or intermittent ischemia. Ohtaki et al49 showed that there was no significant difference between intermittent and single ischemia. However, their model did not mimic the operative setting, since the ischemic times were three 1-hour episodes or a single 3-hour insult. Spetzler and colleagues,50 using a baboon model, demonstrated that short 10-minute periods of repeated occlusions were no different from single occlusions. Finally, the present report provides contradictory data on brain pHi and NADH fluorescence. An interesting observation in these experiments was that the PaCO2 response declined as the number of reperfusions increased. This could be explained in part by a weakening of vessel wall tone due to abrupt surges in perfusion pressure.
Accordingly, it may become evident that, at least in focal cerebral ischemia, there are no significant differences between intermittent or straight occlusion with total ischemic times that approximate the neurosurgical setting. It therefore may prove more valuable to examine the effects of potential neuroprotective agents within the experimental paradigms of single or intermittent reperfusion to better design techniques that provide intraoperative cerebral protection.
| Selected Abbreviations and Acronyms |
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| Acknowledgments |
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| Footnotes |
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Received September 22, 1994; revision received April 17, 1995; accepted May 10, 1995.
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