(Stroke. 1995;26:1634-1638.)
© 1995 American Heart Association, Inc.
Articles |
Brain Temperature Modulations During Global Ischemia Fail to Influence Extracellular Lactate Levels in Rats
From the Cerebral Vascular Disease Research Center, Department of Neurology, University of Miami (Fla) School of Medicine.
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Abstract |
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Background and Purpose While brain tissue lactate increases during cerebral ischemia and is known to be important in the pathogenesis of ischemic brain injury, patterns of extracellular lactate accumulation have been less well characterized, and the influence of brain temperature has not been previously investigated. Mild brain temperature modulations are known to affect the outcome of ischemia dramatically. This study examined changes of extracellular lactate during and after global cerebral ischemia, in which intraischemic brain temperature was held at either 30°C, 37°C, or 39°C.
Methods Halothane-anesthetized fasted male Wistar rats underwent 20 minutes of global cerebral ischemia produced by bilateral carotid artery occlusions plus systemic hypotension (40 to 50 mm Hg). Rectal temperature was maintained at 37°C throughout, and intraischemic brain temperature was held at either 30°C (n=6), 37°C (n=5), or 39°C (n=5). Before and after the ischemic insult, brain temperature was maintained at 37°C in all groups. A microdialysis cannula was implanted in the right dorsolateral striatum and perfused with Ringer's solution. Dialysate samples were collected at 10-minute intervals before, during, and after ischemia and were analyzed for lactate by enzymatic-fluorometric techniques.
Results In all groups, extracellular lactate rose during ischemia and peaked at 10 to 30 minutes of recirculation. Maximal extracellular lactate elevations were sevenfold, eightfold, and eightfold above control in the 30°C, 37°C, 39°C groups, respectively. Significant elevations with respect to control were observed in all groups at 10 to 30 minutes of recirculation. In the 30°C group, these elevations above control were also significant at the 10- and 20-minute ischemic time points (P=.001). At 30 minutes of recirculation, however, lactate levels were lower in the 30°C rats than in the other groups.
Conclusions These data provide evidence that extracellular lactate accumulation is not a crucial determinant of ischemic brain injury. Our results suggest that the increased lactate release during ischemia and the accelerated clearance of lactate during recirculation might contribute in part to the neuroprotection of intraischemic hypothermia.
Key Words: acidosis • hyperthermia • hypothermia • selective vulnerability • rats
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Introduction |
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Lactate, produced by anaerobic glycolysis, has been known to play an important role as a dominant cause of acidosis during ischemia. The higher the lactate accumulation in brain tissue during ischemia, the greater the degree of acidosis and the more severe the resulting ischemic injury in vivo.1 2 3 However, lactic acid has also been reported to have no adverse effect on hypoxic brain tissue, and it is possible that lactate might serve in fact as an energy substrate during hypoxic and aglycemic insults in vitro.4 5
It is well known that the outcome of brain ischemia is sensitive to intraischemic and postischemic temperature changes. Mild hypothermia protects the brain from ischemic injury, and hyperthermia exacerbates damage.6 7 8 9 10 Interestingly, mild hypothermia has been reported to protect the dog brain from an ischemic insult, but without reducing brain tissue lactate levels.11 The present study was designed to investigate the behavior of extracellular lactate in the setting of a global cerebral ischemic insult in which brain temperature during ischemia was held at either normothermic, hypothermic, or hyperthermic levels.
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Materials and Methods |
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General Preparation
Male Wistar rats weighing 250 to 400 g were used after an overnight fast. All animal protocols were approved by the Animal Research Committee of the University of Miami School of Medicine. Anesthesia was induced with 3% halothane, 70% nitrous oxide, and a balance of oxygen. The femoral arteries were cannulated with polyethylene tubing to permit blood pressure measurements and blood gas sampling. Rats were intubated endotracheally and were ventilated mechanically with 0.5% halothane, 70% nitrous oxide, and a balance of oxygen so as to maintain arterial PCO2 and PO2 in the normal range. Animals were immobilized with pancuronium bromide (0.75 mg/kg IV). Both common carotid arteries were exposed via a midline ventral incision and gently separated from the surrounding tissues. Ligatures consisting of polyethylene PE-10 tubing contained within a double-lumen silicone elastomer tubing were passed around each carotid artery. Rectal temperature was maintained at 37.0°C to 37.5°C by means of a warming lamp above the body. Brain temperature was measured with a sterile 33-gauge thermocouple implanted stereotaxically into the left central striatum through a small burr hole drilled in the skull; brain temperature was maintained (at the levels specified below) by means of a warming lamp and a cooling fan (delivering liquid nitrogen vapor) placed above the rat's head.
Microdialysis Procedure
The rat's head was placed in a stereotaxic head
holder. The skull was exposed, and a burr hole was drilled according to
the stereotaxic coordinates for the striatum (1.0 mm
anterior, 3.0 mm lateral to bregma). A microdialysis probe, mounted on
a probe clip and carrier, was lowered into the right dorsolateral
striatum (5.5 mm ventral to dura). The probe was perfused with modified
Ringer's solution at a flow rate of 2 µL/min by means of a
microinfusion pump (Carnegie Medicin). A 2-hour stabilization period
was then allowed. Three 10-minute baseline samples of dialysate were
then collected in an ice bath. At 2.5 hours after probe insertion, the
ischemic insult was initiated. Samples of microdialysis
perfusate were collected at 10-minute intervals continuing throughout
the ischemic and recirculation periods. During the
recirculation period, samples were collected during the first three
10-minute epochs and the last 10-minute epoch of the first hour and
during the last 10 minutes of the second and third hours. These samples
were kept on ice during the collection procedure and were subsequently
frozen and stored at -20°C until analysis.
Production of Cerebral Ischemia
Transient global forebrain ischemia was induced by the
method of bilateral carotid artery occlusions plus systemic
hypotension. First, blood was gradually withdrawn into a heparinized
syringe to reduce systemic blood pressure to 45 to 50 mm Hg. The
carotid ligatures were then tightened bilaterally, and mean blood
pressure was held at 45 to 50 mm Hg by controlled exsanguination.
After 20 minutes of cerebral ischemia, the carotid ligatures
were removed, and the warmed shed blood was reinjected to restore
systemic blood pressure to normal.
Three groups of rats were studied, in which brain temperature during ischemia was held at three different levels: (1) normothermia (intraischemic brain temperature 36.5°C to 37.0°C, n=5); (2) hyperthermia (intraischemic brain temperature 39.0°C to 39.5°C, n=5); and (3) hypothermia (intraischemic brain temperature 29.5°C to 30.0°C, n=6). Before ischemia and during the recirculation period, brain temperature was held at 36.5°C to 37.0°C in all groups.
Assay of Lactate in the Microdialysis Perfusate
Lactate was measured by direct fluorometric assay, with special
precautions taken to avoid contamination. Ten microliters of
microdialysate was added to a buffer of the following constitution
(mmol/L): sodium carbonate 200; hydrazine 50, and diphosphopyridine
nucleotide 0.3. The reaction was initiated by adding 50
µg/mL lactic dehydrogenase. At the end of 45 minutes, samples were
read in a fluorometer (excitation, 360 nm; emission, 460 nm) and the
results plotted against simultaneously run standards.
Dialysate blanks were run at the beginning and end of each
determination. Standards were calibrated by spectrophotometric
techniques with an extinction coefficient of 6.22
cm2/µmol. Lactate concentrations in the dialysate
were not corrected for recovery fraction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The physiological variables are summarized in Tables 1
and 2. There were no
important intergroup differences with respect to arterial
blood pressure or blood gases. Whole-blood glucose levels were similar
in the three animal groups before ischemia, but there was a
trend toward lower blood glucose in the hyperthermic group compared
with the other rat groups during early recirculation. Whole-blood
lactate levels were elevated in all animal groups after 5-minute
postischemic recirculation, but these levels fell by more
than fourfold within the first hour of recirculation.
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The Figure shows patterns of extracellular lactate
accumulation in the three temperature groups. Extracellular lactate
levels rose during ischemia in all groups and peaked during the
early recirculation period. Maximal extracellular lactate elevations
were sevenfold, eightfold, and eightfold above control at
intraischemic temperatures of 30°C, 37°C, and 39°C,
respectively. These elevations were all significant with respect to
control at 10 to 30 minutes of recirculation. In the group with
intraischemic hypothermia (30°C), significant elevations
were noted at the 10- and 20-minute ischemic time points as
well. Repeated-measures ANOVA revealed a highly significant
within-subjects effect for time (F=40.9; df=7,91;
P=.0001). There was also a significant interaction of time
and temperature (P=.001). However, no overall
between-subjects effects were noted for temperature (F=0.54;
df=2,13; P=NS). Univariate post hoc
tests that used the Bonferroni procedure revealed significant
differences between the 30°C and 37°C groups and between the 30°C
and 39°C groups at the 30-minute recirculation time point.
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Significant increases in lactate
above corresponding control level. |
Discussion |
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The results of the study indicate that there is a substantial rise in extracellular lactate concentration as measured by intrastriatal microdialysis during and after 20 minutes of global ischemia in the rat. These elevations, however, were largely independent of intraischemic brain temperature, although the pattern of lactate elevation proved to be somewhat different among the three temperature groups of the study. Major increases in extracellular lactate were not observed during ischemia itself but rather during the early postischemic recirculation period. By contrast, tissue lactate levels are known to increase rapidly during ischemic insults.12 Thus, it is likely that extracellular lactate measured by microdialysis is derived from brain tissue stores. The brain's lactate transport system is an energy-independent and relatively high-capacity system for the release of endogenous lactate by facilitated diffusion, having a Vmax of approximately 44 nmol/mg protein per minute. Accordingly, extracellular and intracellular lactate can equilibrate rather rapidly under conditions of normal or stressed metabolism.13 Thus, the gradual rise in extracellular lactate in this study would suggest that the egress of lactate from brain cells may be rate-limited during ischemia.
Rats in the hypothermic group of the present study tended to attain their highest extracellular lactate levels during ischemia and the first 10 minutes of the recirculation period but had lower lactate levels after 20 to 30 minutes of recirculation. It is known that mild hypothermia is strongly neuroprotective and that mild hyperthermia worsens ischemic outcome.6 As the extent of extracellular lactate accumulation during and after ischemia was largely independent of brain temperature, our results thus suggest that extracellular lactate accumulation in the brain is not a crucial determinant of differences in ischemic outcome as related to brain temperature and that the neuroprotective influence of hypothermia cannot be attributed to the attenuation of cerebral lactate accumulation during ischemia.
Our laboratory has previously reported alterations of regional brain
tissue metabolites in control rats as well as in animals subjected to
global and focal ischemic insults (see Reference 14 for summary
and review). In studies in which preischemic brain glucose
levels averaged 1.5 to 2.2 µmol/g, corresponding average control
levels for brain lactate in these series were 0.8 to 1.1
µmol/g.15 16 17 In contrast, after 1 hour of complete
ischemia at normal body temperature, neocortical tissue lactate
levels rose from control values of approximately 1 µmol/g to levels
exceeding 11 µmol/g, associated with virtually total depletion of
brain glucose and glycogen stores.14 Data from Wistar rats
subjected to 20 minutes of global forebrain ischemia with
cranial temperature regulated at either 30°C, 36°C, or 39°C
during the ischemic insult are shown in Table 3,
which summarizes cortical and striatal tissue lactate levels measured
by enzymatic-fluorometric techniques.14 Table 3 also
provides intraischemic values of tissue lactate under
conditions of brain hypothermia, normothermia, or hyperthermia
measured in an earlier study.6 These data reveal that
there were no significant differences among temperature groups at any
recirculation time point studied (30 minutes, 1 hour, 4 hours). In
contrast, altered intraischemic brain temperature did
influence recovery of ATP and the sum of tissue adenylates.
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Lactate can be utilized as an energy substrate under ischemic or hypoglycemic conditions.4 Metabolism of lactate accumulated in the brain during ischemia can contribute to subsequent recovery of cellular ion transport and electrical activity for a short period of time in the absence of glucose resupply.18 Since there was a trend in the present study for intraischemic hypothermia to accelerate the clearance of extracellular lactate, it is conceivable that this was mediated by an increase in lactate metabolism and hence increased energy supply to postischemic neurons, contributing to hypothermic neuroprotection.
Although lactate has been suspected to be a prime factor contributing to brain injury during and after ischemia, and a linear relationship has been described between lactate accumulation in tissue and ischemic outcome,19 20 21 22 23 there is nonetheless no evidence to suggest that lactate itself is harmful. Indeed, neurons exposed to 20 mmol/L lactate at normal pH for up to 6 hours were undamaged in vitro.24 In fact, lactate may have a protective role in preventing cell death mediated by calcium overload under ischemic-type conditions by inhibiting the rise in intrasynaptosomal calcium.25
While there is no evidence to suggest that lactate itself is harmful to tissue, the low pH associated with lactate accumulation can directly disrupt cell membranes, leading rapidly to cell death within as little as 30 minutes. With exposure to more moderate acidity, cell death occurs with a latency of as long as 24 to 48 hours; this delayed lethal injury can be attenuated by postinjury hypothermia.24 Hypothermia has been reported to reduce cerebral intracellular acidosis significantly.26 Accelerated lactate metabolism would be expected to ameliorate deleterious lactic acidosis, and the present results suggest that intraischemic hypothermia may possibly tend to act in this manner.
Evidence suggests that most of the lactate released by brain tissue in vitro is derived from astrocytes.27 28 Astrocytes contribute 6.3 times as much lactate as neurons under normal conditions, and this increases to 7.7-fold in normoglycemic ischemia and to 12.2-fold with hyperglycemic ischemia. It is estimated that astrocytes account for more than 90% of lactate production. Astrocytes continue to release lactate even though glucose is completely removed from the medium, and the lactate released from astrocytes equilibrates quickly with all central nervous system compartments by transport via a monocarboxylic acid carrier and passive diffusion. We hypothesize that the extracellular lactate measured in ischemia in the present study may derive mainly from astrocytes, which release lactate in an effort to support neuronal function during ischemia. Intraischemic hypothermia may stimulate glial metabolism during ischemia to accelerate lactate release.
To summarize, our results suggest that extracellular lactate accumulation of itself is not a crucial determinant of ischemic brain injury. We suggest that lactate might serve as an energy supply to promote neuronal survival in ischemia provided that the associated intracellular acidosis can be promptly ameliorated. Increasing lactate release during ischemia and accelerating lactate clearance during recirculation might tend to contribute in part to the neuroprotective effect of intraischemic hypothermia.
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Acknowledgments |
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This study was supported by National Institutes of Health grant NS 05820. Helen Valkowitz helped to prepare the typescript.
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Footnotes |
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Reprint requests to Baowan Lin, MD, Department of Neurology (D4-5), University of Miami School of Medicine, PO Box 016960, Miami, FL 33101.
Received February 22, 1995; revision received May 30, 1995; accepted June 6, 1995.
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References |
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