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(Stroke. 1996;27:1592-1602.)
© 1996 American Heart Association, Inc.
Articles |
Effects of Preischemic Hyperglycemia on Brain Damage Incurred by Rats Subjected to 2.5 or5 Minutes of Forebrain Ischemia
the Laboratory for Experimental Brain Research, Lund University (P.-A.L., T.K., M.S., B.K.S.), Sweden, and the Institute of Neurobiology, Slovak Academy of Science (T.K.), Kosice, Slovak Republic.
Correspondence to Bo K. Siesjo, Laboratory for Experimental Brain Research, Wallenberg Neuroscience Center, University Hospital, S-221 85 Lund, Sweden.
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Abstract |
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Background and Purpose The objective of this study was to explore whether preischemic hyperglycemia, which is known to aggravate brain damage due to transient global or forebrain ischemia of intermediate duration (10 to 20 minutes), increases the density of selective neuronal necrosis, as observed primarily in the CA1 sector of the hippocampus after brief periods of forebrain ischemia in rats (2.5 and 5 minutes).
Methods Anesthetized rats were subjected to two-vessel forebrain ischemia of 2.5- or 5-minute duration. Normoglycemic or hyperglycemic rats were either allowed a recovery period of 7 days for histopathological evaluation of neuronal necrosis in the hippocampus, isocortex, thalamus, and substantia nigra or were used for recording of extracellular concentrations of Ca2+ ([Ca2+]e), K+, or H+, together with the direct current (DC) potential.
Results Ischemia of 2.5- or 5-minute duration gave rise to similar damage in the CA1 sector of the hippocampus in normoglycemic and hyperglycemic groups (10% to 15% and 20% to 30% of the total population, respectively). However, in hyperglycemic animals subjected to 2.5 minutes of ischemia, CA1 neurons never depolarized and [Ca2+]e did not decrease. In the 5-minute groups, the total period of depolarization was 2 to 3 minutes shorter in hyperglycemic than in normoglycemic groups. This fact and results showing neocortical, thalamic, and substantia nigra damage in hyperglycemic animals after 5 minutes of ischemia demonstrate that although hyperglycemia delays the onset of ischemic depolarization and hastens repolarization and extrusion of Ca2+, it aggravates neuronal damage due to ischemia.
Conclusions These results reinforce the concept that hyperglycemia exaggerates brain damage due to transient ischemia and prove that this exaggeration is observed at the neuronal level. The results also suggest that the concept of the duration of an ischemic transient should be qualified, particularly if ischemia is brief, ie, <10 minutes in duration.
Key Words: cerebral ischemia • depolarization • hippocampus • histopathology • hyperglycemia
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Although it has been established beyond doubt that preischemic hyperglycemia aggravates damage due to transient global or forebrain ischemia (for reviews, see References 1 and 2), several questions demand an answer. The first concerns the mechanisms involved. It now appears likely that hyperglycemia acts by exaggerating the reduction of pHe and pHi. Thus, aggravation of brain damage incurred as a result of 10 minutes of forebrain ischemia in rats occurs over a narrow range of plasma glucose concentrations (12 to 16 mmol/L), and results concerning seizure incidence in these animals suggest a critical pHe threshold around 6.4.3 These results are compatible with a narrow range of pH values over which damage is exaggerated, eg, by iron-catalyzed free radical production.4
The second question concerns the cellular and subcellular targets involved. In animals subjected to 
10 minutes of ischemia, hyperglycemia typically causes pannecrotic lesions and exaggerated edema.5 6 7 At least after long periods of ischemia, this may in part reflect damage to nonneuronal structures, such as glial cells or microvessels.8 9 10 It also seems established that a major subcellular target is the mitochondrion.11 12 13
The present series of experiments was designed to provide an answer to the question, Is selective neuronal necrosis exaggerated by enhanced intraischemic acidosis, in the likely absence of damage to glial cells or microvessels? To provide answers to this question, we induced forebrain ischemia of only 2.5-minute duration in normoglycemic and hyperglycemic rats and evaluated CA1 damage after 7 days of recovery. Somewhat unexpectedly, normoglycemic and hyperglycemic subjects showed a similar extent of CA1 neuronal necrosis, ranging between 10% and 20% of the total neuronal population. What was remarkable, though, was that hyperglycemic animals showed neither signs of cellular depolarization nor a decrease in the extracellular calcium concentration in the dorsal hippocampus.14 These results obviously challenge the postulate that membrane depolarization and calcium influx are what trigger ischemic cell death.
In the present article, we give a full account of experiments with 2.5 minutes of ischemia and provide data on changes in pHe as well as in [Ca2+]e, [K+]e, and DC potential and extend the research to 5 minutes of ischemia in normoglycemic and hyperglycemic animals. The results obtained provide additional information about acidosis-related neuronal necrosis and challenge conventional postulates about mechanisms involved in ischemic brain damage. Furthermore, they raise the question of how the duration of an ischemic insult should be defined.
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Animals and Operative Techniques
The experiments were performed on male, overnight-fasted Wistar rats of a special pathogen-free strain, weighing 270 to 340 g (Møllegaard's Breeding Center, Copenhagen, Denmark). Anesthesia was induced with 3.5% halothane in N2O/O2 (70:30). The animals were intubated and connected to an animal respirator, and the halothane concentration was reduced to 1% to 1.5% during operation. A tail artery and a tail vein were cannulated for blood pressure recordings, blood sampling, and glucose or drug infusions. A soft silicone elastomer catheter was inserted into the inferior caval vein through the jugular vein on the right side to manipulate blood pressure during the induction of ischemia. The common carotid arteries on both sides were isolated and encircled with loose ligatures for later clamping. After operation, the halothane concentration was lowered to 0.5% and the ventilation and O2 supply were adjusted to give an arterial PaCO2 of 4.66 to 5.32 kPa (35 to 40 mm Hg) and a PaO2 of close to 13.30 kPa (100 mm Hg). Heparin (90 IU/kg) was given before the first blood gas measurement. The animals were paralyzed with vecuronium bromide (2 mg/h) during the experiment. Core temperature was maintained at 37°C by a homeothermic blanket control unit, and head temperature was maintained at 37°C by lamp heating. Animals used for ion measurement were placed in a Faraday cage on a plastic table, and the head was fixed inside a heating box to maintain the head temperature close to 37°C during the experiment. Another heating box was used to calibrate the electrodes under conditions similar to those encountered during measurements (for details, see Reference 15). Plasma glucose concentration was measured with a Beckman Glucose Analyzer 2 (Beckman Instruments Inc). Arterial blood gases (PaCO2 and PaO2) and pH were measured with a standard microsample blood gas monitor (ABL 300, Radiometer).
After termination of ischemia, 0.5 mL of a 0.6 mol/L NaHCO3 solution was given intravenously to counteract systemic acidosis, and the halothane supply was discontinued after reperfusion in the animals used for histopathological studies and continued for 15 minutes in those used for ion measurements. When they had regained spontaneous breathing, the animals used for histological studies were extubated and disconnected from the respirator. They were then housed in cages with free access to tap water and pellet food and were perfusion fixed after 7 days of recirculation.16 17 All animal treatments followed the guidelines of the National Institutes of Health (Guide for the Care and Use of Laboratory Animals) and were approved by the Ethical Committee for Laboratory Animal Experiment at the University of Lund.
Microelectrode Measurements
[Ca2+]e, [K+]e, and pHe were measured by double-barreled glass microelectrodes that were pulled from two aligned glass capillaries in a vertical gravitational puller. The tip diameter of the microelectrodes was 4 to 6 µm. The barrels intended for the ion-sensitive liquid membrane were silanized in dimethyldichlorosilane (Fluka AG) vapor for 2 minutes. A 1-mm column of Ca2+ cocktail (Fluka AG) was filled at the tip of the silanized barrel with 0.1 mol/L CaCl2 used as an inner reference solution for calcium electrodes; a K+ cocktail (Fluka AG) and 0.15 mol/L KCl were used for potassium electrodes; and a H+ cocktail (Fluka AG) and Tris-HCl (0.05 mol/L, pH 7.0) were used for pHe electrodes. The reference barrels were filled with 0.15 mol/L NaCl for [Ca2+]e, [K+]e, or pHe electrodes. The calcium electrode was calibrated in calibration solutions containing 0.1, 0.5, 1.0, and 5 mmol/L CaCl2 (for construction and calibration, see References 18 and 19); the potassium electrode was calibrated in solutions containing 2, 10, 50, and 80 mmol/L KCl19 20 ; and the pHe electrode was calibrated in pH 6.4, 7.0, and 7.4 phosphate buffers and in an artificial cerebrospinal fluid (for details of construction and calibration, see References 21 and 22). Connection between solutions and high-input impedance amplifiers was made by use of Ag/AgCl wires. The [Ca2+]e, [K+]e, or pHe was obtained by differential recording between the ion-sensitive and reference barrels. DC potential (or reference potential) was recorded between the reference barrel and an external ground Ag/AgCl-agar electrode placed subcutaneously on the back of the body. The slopes of calcium, potassium, and pHe electrode responses were 26 to 30 mV, 50 to 60 mV, and 50 to 55 mV per decade change of ion concentration or per pH unit, respectively. The response time of electrodes was 
1 second.
[Ca2+]e and pHe were recorded in both the isocortex and the hippocampal CA1 region. Extracellular potassium was only recorded in the hippocampus. The ion-sensitive microelectrodes were placed though a hole drilled on the cranium 3 mm lateral and posterior and 0.7 mm below the bregma for measurements in the frontoparietal cortex, and 2.2 mm lateral, 3.8 mm posterior, and 2.15 mm below the bregma for recordings in the stratum radiatum of the CA1 region. The position of the microelectrode tip was controlled in five animals by releasing dye and checking under the microscope after perfusion and sectioning of the brains. The tip was positioned in the stratum radiatum of the CA1 region.
Experimental Protocol
Two brief periods of forebrain ischemia, nominally 2.5 and 5 minutes in duration, were used in the present experiments to explore whether selective neuronal necrosis in the CA1 sector of the hippocampus was aggravated by enhanced acidosis after such brief periods of ischemia. Preischemic hyperglycemia was induced by infusion of a 25% glucose solution for 30 minutes before induction of ischemia (2.3 mL/h), elevating plasma glucose concentration to 20 mmol/L. Normoglycemic animals were infused with the same volume of Krebs-Henseleit solution for an identical period.
One hundred eleven rats were divided into two major groups. In the first group, 41 animals were subdivided into four subgroups (n=8 to 12 in each group) for histopathological analysis. In the second group, 68 animals were used for [Ca2+]e, [K+]e, and pHe recordings. [Ca2+]e (n=34) and extracellular pHe (n=34) were recorded in both the isocortex and the stratum radiatum of the hippocampal CA1 region in all experimental groups (n=4 to 6 in each), whereas [K+]e was only recorded in 2 animals in the CA1 sector to confirm that there was no membrane depolarization or massive K+ efflux during the 2.5 minutes of ischemia in hyperglycemic subjects. For details of the groups, please see Tables 1
and 2.
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Forebrain ischemia of 2.5- and 5-minute duration was induced in consecutive order by reduction of blood pressure to a range of 5.32 to 6.65 kPa (40 to 50 mm Hg) by bleeding, followed by bilateral carotid artery clamping.23 After clamping, MABP rose spontaneously to >13.30 kPa (100 mm Hg). The beginning of the period of ischemia was defined as the time when the blood pressure was rereduced to and maintained at 5.32 to 6.65 kPa (40 to 50 mm Hg) by further withdrawal of blood from the jugular vein. The time from bilateral common carotid artery clamping to the time considered to represent the beginning of ischemia was 0 to 60 seconds (see Reference 23). The EEG, recorded from the skull, became isoelectric 10 to 30 seconds after clamping. The end of the period of ischemia was defined as the time when the carotid clamps were removed and blood was reinfused. This procedure, ie, the removal of the carotid clamps and the infusion of blood to yield an MABP of 13.30 kPa (100 mm Hg), usually took 10 seconds.
[Ca2+]e, [K+]e, pHe, and DC potential were recorded continuously for 30 minutes before ischemia, during 2.5 or 5 minutes of ischemia, and for an additional 15 minutes after the start of recirculation. After 7 days of recovery, the animals used for histopathological studies were reanesthetized with halothane and perfusion fixed with 4% formaldehyde. The brains were removed, dehydrated, embedded in paraffin, and sectioned coronally at 5 µm and stained with a combination of celestine blue and acid fuchsin.24
Quantification of Brain Damage
Quantification of damaged neurons, evaluated in a blinded manner, was performed by direct visual counting of acidophilic neurons at a magnification of x400. Neuronal damage in the subiculum and CA1 of the hippocampus and in two isocortex structures (parietal and cingulate cortex) was counted in one coronal section at the level of the bregma minus 3.8 mm and presented as the average number of dead neurons in each hemisphere. Damage in the ventroposterior nucleus of the thalamus and in the pars reticulata of the substantia nigra was scored on a four-grade scale on which grade 0 meant no observed damage; grade 1, <10% damaged neurons; grade 2, 11% to 50% damaged neurons; and grade 3, >50% damaged neurons.
Statistics
Physiological parameters and histopathological data in the CA1 sector of the hippocampus were analyzed by two-factor ANOVA followed by Scheffe's test. Histopathological data in the parietal cortex, cingulate cortex, substantia nigra, and thalamus were analyzed by nonparametric Mann-Whitney U test. A paired Student's t test was used to compare intraischemic and postischemic pHe levels at different recirculation time points with preischemic pHe control levels in the same animal.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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We will describe the histopathological outcome of forebrain ischemia of 2.5- and 5-minute duration and raise the question about crucial pathophysiological events. In this analysis, we will reexamine the concept of ischemia duration and correlate this definition to changes in DC potential and [Ca2+]e in both the isocortex and the CA1 sector of the hippocampus.
Tables 1 and 2
describe the groups assembled and give physiological parameters (and variables) in animals subjected to 2.5 or 5 minutes of ischemia. MABPs were in the range of 14.63 to 15.96 kPa (110 to 120 mm Hg), body temperature was 37°C, PaCO2 was 5.32 kPa (40 mm Hg), PaO2 exceeded 13.30 kPa (100 mm Hg), and pH was close to 7.4. Normoglycemic animals had plasma glucose concentrations of 5 mmol/L, and hyperglycemic animals had values close to 20 mmol/L.
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Histopathology
Results concerning hippocampal CA1 damage are summarized in Fig 1, and representative photomicrographs are shown in Fig 2. As already reported,14 2.5 minutes of ischemia produced similar hippocampal damage in the two groups (10% to 20% of total CA1 cell population). With prolongation of the ischemia to 5 minutes, the number of necrotic neurons was doubled in both groups, but because of a large interanimal variability, the difference between normoglycemic and hyperglycemic animals was not significant. In the subiculum, the damage was 20% and 50% after 2.5 and 5 minutes of ischemia, respectively (data not shown). To explore whether hyperglycemia aggravates neuronal damage under the same conditions in terms of total duration of the calcium transient, the 2.5-minute normoglycemic group was compared with the 5-minute hyperglycemic group because the duration of the calcium transient was the same in the two groups (4 to 5 minutes; see below). In this comparison, the latter group had a significantly higher number of necrotic neurons (P<.01).
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, normoglycemic animals; 
, hyperglycemic animals. **P<.01 by two-factor ANOVA followed by Scheffe's test.
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In the 2.5-minute ischemia group, neuronal necrosis was not observed outside the CA1 sector of the hippocampus. However, after 5 minutes of hyperglycemic ischemia, the majority of animals showed frontoparietal cortex damage, and about half of the animals had unequivocal damage in the cingulate cortex, the substantia nigra, or the thalamic nuclei (Fig 3). Examples of such damage are illustrated in Fig 4. As can be observed, the lesions had the characteristics of selective neuronal necrosis, while involvement of glial cells and microvessels could not be observed.
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DC Potential Shifts and Calcium Transients
Fig 5 illustrates [Ca2+]e transients in the isocortex and hippocampus during 2.5 minutes of ischemia in normoglycemic and hyperglycemic animals. In the isocortex of normoglycemic animals (Fig 5A), induction of ischemia was followed within an interval of 30 to 40 seconds by a precipitous decrease in [Ca2+]e to values of 0.1 to 0.2 mmol/L, reflecting cellular uptake of calcium. After reperfusion and a delay of 60 to 90 seconds, [Ca2+]e gradually increased, reflecting resumption of Ca2+ extrusion. In the hippocampus, changes in [Ca2+]e during ischemia were similar, with control values being reached in 15 minutes in both structures.
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The corresponding [Ca2+]e transients in hyperglycemic animals are shown in Fig 5B. As reported previously,19 hyperglycemia delayed the onset of depolarization and calcium influx in the isocortex and accelerated Ca2+ extrusion after recirculation; nonetheless, 2.5 minutes of ischemia was sufficient to allow depolarization and calcium influx. In contrast, no such depolarization/Ca2+ influx was observed in the hippocampus (see also Reference 14).
The results shown in Fig 5 and Table 3 demonstrate the following: First, normoglycemic and hyperglycemic rats had similar preischemic [Ca2+]e values, and the values were similar in the isocortex and hippocampus. Second, in all normoglycemic animals, [Ca2+]e was reduced to 15% of control or lower at the end of the ischemic periods. Values for the CA1 sector were as low or even lower than in the isocortex. In the 2.5-minute hyperglycemic group, the isocortex showed a value of 25% of control at the end of the ischemic period, whereas the hippocampus had an unchanged (or increased) value. In the 5-minute groups, [Ca2+]e was uniformly low. In all groups, [Ca2+]e returned to control values after 15 minutes.
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In summary, preischemic hyperglycemia delayed the precipitous reduction in [Ca2+]e and the DC potential shift induced by ischemia. The suggested rise in [Ca2+]e after 2.5 minutes of ischemia could have reflected a moderate decrease in extracellular fluid volume. In support of this conclusion, the two rats with [K+]e recordings showed slowly increasing [K+]e during ischemia, with 
[K+]e values of 1.9 and 2.1 mmol/L, respectively.
Latency to Depolarization and Duration of Depolarization Periods
The results of the present study raise a question concerning the definition of the period of ischemia when the nominal duration is 
5 minutes. Fig 6 gives the latency to ischemic depolarization in the 2.5- and 5-minute groups. In the isocortex, the latency increased from 20 to 60 seconds in normoglycemic rats and from 90 to 140 seconds in hyperglycemic rats, ie, hyperglycemia prolonged the predepolarization period by >1 minute. In the hippocampus, the prolongation was even more marked because hyperglycemia delayed depolarization by 2 minutes.
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The difference between the normoglycemic and hyperglycemic groups becomes even more pronounced if one also takes into account the fact that hyperglycemia reduces the lag between reperfusion and membrane repolarization and the start of Ca2+ reextrusion. In Fig 7, the total depolarization period was calculated as the time between the sudden DC potential negativity and the almost equally rapid positive deflection. In ischemia of nominal 2.5-minute duration, there was a difference between normoglycemic and hyperglycemic animals of almost 3 minutes in the isocortex and of >3 minutes in the hippocampus. In the 5-minute ischemia groups, two animals had relatively long total depolarization periods. For that reason, mean differences between normoglycemic and hyperglycemic groups were only 2 minutes.
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Extracellular pH Changes
Fig 8 illustrates a typical recording showing how pHe changes in the hippocampal CA1 area in a normoglycemic and a hyperglycemic rat during 5 minutes of ischemia. As illustrated in Fig 8, pHe started to fall when bleeding from the jugular vein was begun and then continued to decrease after clamping, reaching values of 6.7 in the normoglycemic rats and 6.3 in the hyperglycemic rats. After recirculation and repolarization, pHe showed an initial fast recovery, followed by a slow return toward control values.
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Fig 9 shows group data. In normoglycemic animals subjected to 2.5 minutes of ischemia, pHe in the isocortex and hippocampus decreased to a range of 6.7 to 6.9. In hyperglycemic animals, the values were lower (6.4 to 6.5). Recovery was gradual and seemingly slightly slower in the hippocampus. After 5 minutes of hyperglycemic ischemia, the pHe values reached were similar, suggesting that acidosis was already maximal after 2.5 minutes. In normoglycemic animals, pHe was again somewhat lower in the hippocampus than in the isocortex, but after 5 minutes, no such trend existed.
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Discussion |
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The main question we posed when designing the present experiments was, Is neuronal necrosis exaggerated by preischemic hyperglycemia? This was the reason we studied ischemic periods of 2.5 and 5 minutes, periods that are usually associated with <50% damage in the CA1 sector and little (if any) neuronal necrosis in other regions. On the basis of conventional criteria for defining the duration of ischemia, we can conclude that hyperglycemia fails to exaggerate CA1 damage but that it triggers scattered extrahippocampal damage in animals subjected to 5 minutes of ischemia.
This conclusion could be of questionable scientific value, however, because an ischemic period of 2.5 minutes in hyperglycemic animals usually did not lead to signs of CA1 cell depolarization or to a fall in [Ca2+]e. Furthermore, in animals subjected to 5 minutes of ischemia, the total depolarization period was less than half as long in hyperglycemic animals as that in normoglycemic animals. Thus, the question must be raised: Why was there CA1 damage at all in hyperglycemic animals subjected to 2.5 minutes of ischemia? Furthermore, why was damage equal after 5 minutes of ischemia in normoglycemic and hyperglycemic rats, although the former showed depolarization after 1 minute and the latter after 3 minutes?
Potential methodological pitfalls should be discussed, primarily those arising from measurements of [Ca2+]e, [K+]e, and pHe in a single site. Theoretically, such measurements could miss depolarizations occurring in other parts of the CA1 sector. This is not likely, however. First, in ischemic tissue, any local depolarization is apt to cause a spreading depression in the entire CA1 sector.25 Second, our recent data demonstrate that in hyperglycemic animals, the ATP concentration does not fall below 50% of control during 2.5 minutes of ischemia (J. Folbergrova, PhD, P.-A. Li, MD, H. Uchino, MD, M.-L. Smith, PhD, B.K. Siesjo, MD, PhD; unpublished data, 1996). Experiments with the isocortex demonstrate that depolarization first occurs when the overall ATP concentration is reduced to 30% to 40%.26
There are two reasons why preischemic hyperglycemia can prolong the time before depolarization occurs during ischemia and hasten repolarization during recirculation. First, an increased tissue glucose concentration enhances the production of ATP, which can be used for ion transport.27 28 29 Second, enhanced acidosis reduces the rate of influx of Ca2+ and Na+ into cells during ischemia,30 thereby altering the leak-pump relationship for ion flux.
The present results suggest that we should scrutinize the term "duration of ischemia" and attempt to consider factors that are critical when ischemia leads to irreversible brain damage. Clearly, if CBF is reduced to only 30% or 40% of control, be it for 5 or 60 minutes, we would accept results demonstrating that no brain damage is incurred. In this case, it is not meaningful to discuss the duration of ischemia even if the reduction in CBF is associated with symptoms of cell distress. However, if the ischemia is severe (for example, with a CBF of <10% of control), 3 to 5 minutes of ischemia leads to neuronal necrosis not only in the gerbil, but also in the rat.17 31 It is in the latter situation, ie, with a severe reduction in CBF, that we encounter problems in defining the duration of ischemia. The problem is trivial if ischemia duration is 15 minutes, but it is pertinent if one studies ischemia of <10 minutes' duration. The question at stake is, What factor(s) in the ischemic cascade is/are the crucial one(s)?
The present results show that neither cell depolarization nor translocation of Ca2+ from extracellular to intracellular fluids is required for CA1 damage to be incurred. What, then, gives rise to the neuronal necrosis observed? A study parallel to the present one demonstrated that 2.5 minutes of ischemia in hyperglycemic subjects was accompanied by a decrease in tissue ATP concentration toward 50% of control, with corresponding increases in ADP and AMP concentrations and a rise in lactate content to 13 mmol/kg (J. Folbergrova et al, unpublished data, 1996). This information and the present results showing a decrease in pHe to 6.5 identify a decrease in phosphorylation potential and lactic acidosis as possible triggering factors for delayed cell death. It has also been surmised32 and indeed demonstrated33 that intracellular calcium rises long before [Ca2+]e is reduced, suggesting release of Ca2+ from intracellular binding/sequestration sites. In this context, it is of interest that Shen et al34 observed neuronal damage in cultured hippocampal slices when pHe was lowered to only 6.62 for 30 minutes. Interestingly, cell damage was markedly reduced in the absence of oxygen, suggesting that free radical production was responsible.35
In spite of this, it seems likely that membrane depolarization and a massive influx of Ca2+ are important determinants of the ultimate damage incurred after transient ischemia. But if this is so, how do we define the duration of ischemia? Fig 10 shows data we have compiled from several experiments to demonstrate how induction of ischemia and recirculation are related to changes in EEG, DC potential, and [Ca2+]e in normoglycemic animals in the cortex. From this diagram, one could identify the period of ischemic depolarization as the period between the rapid negative shift to the return of the DC potential toward previous values. Both of these time points can be well demarcated, and they correspond to the rapid efflux-influx of K+. However, the problem then becomes how to define the corresponding Ca2+ transient. Its onset is easy to define, but not its recovery. Thus, although the initial recovery coincides with the (positive) DC potential shift and with K+ influx there follows a secondary, slow return of [Ca2+]e toward control values, suggesting that in terms of duration of the potentially harmful Ca2+-mediated events subsequent to ischemia, normalization is relatively slow.
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, induction of 5 minutes of ischemia; 
, recirculation.It is evident that the duration of ischemia ordinarily will still be defined in terms of the duration of the decrease in CBF to low values; however, any discussion of ischemic duration must take into account the fact that for a given duration of ischemia, as conventionally defined, the duration of membrane "failure" and of massive calcium loading may vary widely, depending on the circumstances.
Siemkowicz and Hansen,27 studying recovery of [Ca2+]e after transient ischemia, suggested that the biphasic recovery of [Ca2+]e reflected the rapid extrusion of calcium available to the plasma membrane transporters, followed by the slow extrusion of the Ca2+ that was released from intracellular sequestration stores, such as the mitochondria.36 37 38 But if this is the case, we have difficulty determining when the ischemic transient has been terminated. Thus, we can pose the question, When have the mitochondria released their Ca2+ load? The blood-brain barrier is very sparingly permeable to Ca2+.38 Therefore, the cell calcium content and, very likely, the mitochondrial calcium content are back to control values when [Ca2+]e has normalized. Because this takes 10 minutes, the duration of the calcium transient is appreciably longer than the nominal periods of ischemia.
The question about the duration of the calcium transient is a pertinent one because as long as the mitochondrial (matrix) content is increased, Ca2+ could conceivably induce pathology, such as an MPT or activation of mitochondrial phospholipase A2, with ensuing damage to the lipid constituents of the inner mitochondrial membrane.39 40 41 Because the tissue has been subjected to an anaerobic/aerobic redox transition, the stage has been set for the enhanced production of ROS and free radical–related damage.42 44 45 In fact, there appears to be an intimate relationship among mitochondrial calcium accumulation, production of ROS, and cell damage of the apoptotic/necrotic type. For example, oxidative stress, in combination with mitochondrial calcium accumulation, is known to open a "megachannel" in the inner mitochondrial membrane, thereby inducing an MPT.45 46 This permeability transition is considered to be detrimental for two reasons: it floods the cytoplasm with calcium, and it causes additional production of ROS. In other systems, oxidative stress is considered to cause the release of calcium from the mitochondria and calcium cycling across their inner membranes.47 48
The relationship among mitochondrial calcium accumulation, production of ROS, and mitochondrial dysfunction has recently become of interest. As mentioned, work on isolated mitochondria has shown that oxidative stress and calcium accumulation trigger the induction of a "megapore" in the inner mitochondrial membrane47 48 ; in all probability, the opening of this pore is synonymous with the induction of an MPT in other settings.41 The induction of an MPT is suppressed in a virtually specific way by the immunosuppressant drug cyclosporin A. Richter47 and Richter et al48 have proposed that oxidative stress can cause the release of calcium from mitochondria without prior activation of a large, unspecific membrane conductance. Cycling of calcium across the inner mitochondrial membrane will lead to mitochondria damage. Interestingly, several groups have suggested that reperfusion damage is triggered by a permeability transition of the inner mitochondrial membrane.39 40 41 Studies of mitochondria from many sources demonstrate that the assembly of a proteinaceous megapore in the inner mitochondrial membrane is enhanced by high Ca2+, high Pi, and oxidative stress and retarded by high ADP and low pH.42 Thus, the probability of a permeability transition is highest when reperfusion has caused mitochondrial calcium accumulation and production of ROS and when pH and ADP concentrations have normalized.
Richter47 suggested that oxidative stress with release and cycling of calcium triggered delayed cell death of an apoptotic type. Others49 50 have shown that when apoptosis is induced in rapidly proliferating cells, such as thymocytes, a series of events is sequentially induced that starts with an MPT, continues with Ca2+ release and production of ROS by the mitochondria, and ends with nuclear and plasma membrane damage and cell death. However, the mode of cell death is not necessarily apoptosis, because results obtained in tissue cultures from nonneuronal and neuronal sources suggested that an intense but brief insult (eg, glutamate exposure) or a longer one of moderate severity leads to apoptotic cell death, whereas severe and long-lived insults give rise to necrotic cell death.51
CA1 damage incurred in normoglycemic animals after 7 to 10 minutes of ischemia is dramatically ameliorated by cyclosporin A.52 This finding, plus evidence of the gradual accumulation of calcium in the cells during the postischemic period ( for discussion and references, see References 5353 and 5454 ), suggested that cell death may be triggered by an MPT. It is conceivable that brief periods of ischemia lead to apoptotic cell death by similar mechanisms, ie, by causing primary oxidative damage to plasma membrane, they secondarily lead to cell calcium accumulation and ultimately to mitochondrial calcium overload, with induction of an MPT that has detrimental consequences for cell survival. At present, this deduction is speculative, and we do not know for certain that the scheme suggested applies to damage affecting CA1 cells after 2.5 minutes of ischemia in hyperglycemic animals. However, circumstantial evidence exists that this is so, because our recent data demonstrate that cyclosporin A prevents the CA damage from occurring (J. Folbergrova et al, unpublished data, 1996).
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Acknowledgments |
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This work was supported by the Swedish Medical Research Council (14X-263), the US Public Health Service via the NIH (5 R01-NS-07838), and the Juvenile Diabetes Foundation International.
Received February 21, 1996; revision received May 2, 1996; accepted May 15, 1996.
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References |
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Editorial Comment
Department of Anesthesiology and Critical Care MedicineThe Johns Hopkins UniversityBaltimore, Md
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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Hyperglycemia is well known to enhance necrosis after global cerebral ischemia by a mechanism related to increased tissue lactic acidosis. The acidosis-related injury is not restricted to neurons but also involves ultrastructural changes in astrocytes and endothelium.1R 2R Thus, the enhanced neuronal injury may be secondary to astrocytic and endothelial dysfunction rather than to a direct effect of acidosis on ischemic neurons. In cell culture, acidosis can actually protect neurons by altering the redox state of N-methyl-D-aspartate receptors.
Previous studies in vivo1R 2R 3R have focused on ischemia durations of 10 to 30 minutes. In the present study, the authors examined shorter durations of ischemia in which selective neuronal necrosis was modest, without major involvement of astrocytic or endothelial damage, and in which any augmentation of damage by hyperglycemia should be readily discernible by counting necrotic neurons. With 5 minutes of forebrain ischemia in the rat, the authors found that preischemic hyperglycemia augmented the decrease in extracellular pH but delayed the onset of cell depolarization and the associated decrease in extracellular calcium activity that occurs with massive calcium influx. Neuronal necrosis was evident in the cortex, thalamus, and substantia nigra in many of the hyperglycemic rats but in none of the normoglycemic rats despite the fact that hyperglycemia shortened the duration that cortical cells were depolarized. These results are important because they clearly demonstrate that acidosis can augment ischemic necrosis of neurons.
In CA1 hippocampus, the results were not as straightforward. Neuronal necrosis was evident in normoglycemic rats with as little as 2.5 minutes of ischemia. Hyperglycemia did not significantly augment neuronal necrosis in CA1 after 2.5 or 5 minutes of ischemia, although the duration of cell depolarization was shorter. Remarkably, cell depolarization was not detected in the CA1 region of hyperglycemic rats, yet necrosis still occurred in a portion of the neurons. These results suggest that acidosis can promote ischemic injury even when cells do not fully depolarize and there is not a massive calcium influx. Neuronal injury in this region is known to mature over a period of several days by mechanisms that are still unknown but could involve mitochondrial dysfunction, free radicals, and apoptotic mechanisms. Hyperglycemia accelerates this maturation of injury.4R
The authors make a point that with short ischemic durations, one has to be careful how to define ischemic duration. Perhaps ischemia should be defined as the period of cell depolarization rather than the period of reduced blood flow. However, there is evidence of water and ion influx preceding full depolarization.5R 6R 7R 8R An early, localized increase in intracellular calcium is possible9R and may be sufficient to cause substantial neurotransmitter release or to stimulate signal transduction pathways at a time when the phosphorylation potential is decreasing. Acidosis may interfere with the astrocytic uptake of neurotransmitters, interact with signal transduction pathways, and promote reactive oxygen species that can activate transcription factors in the absence of full depolarization. In the present study, acidosis took an extra 10 to 15 minutes to recover after ischemia in hyperglycemic rats, and acidosis during the early period of reoxygenation could augment free radical damage. All of these mechanisms are speculative but deserve further investigation for understanding how acidosis modifies ischemic neuronal injury.
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Selected Abbreviations and Acronyms |
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Normo indicates normoglycemic rats; Hyper, hyperglycemic rats.
Data are expressed as mean±SD. Data in the 2.5- and 5-minute ischemia groups were analyzed separately.
*P<.0001 against the other three subgroups in the 2.5-minute ischemic group by two-factor ANOVA followed by Scheffe's test.
P<.01 compared with the normoglycemic group in the cortex by unpaired Student's t test.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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1R. Paljarvi L. Brain lactic acidosis and ischemic cell damage: a topographic study with high-resolution light microscopy of early recovery in a rat model of severe incomplete ischemia. Acta Neuropathol (Berl).. 1984;64:89-98.[Medline] [Order article via Infotrieve]
2R. Paljarvi L, Rehncrona S, Soderfeldt B, Olsson Y, Kalimo H. Brain lactic acidosis and ischemic cell damage: quantitative ultrastructural changes in capillaries of rat cerebral cortex. Acta Neuropathol (Berl).. 1983;60:232-240.
3R. Li P-A. Shamloo M, Smith M-L, Katsura K, Siesjo BK. The influence of plasma glucose concentrations on ischemic brain damage is a threshold function. Neurosci Lett.. 1994;177:63-65.[Medline] [Order article via Infotrieve]
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6R. Decanniere C, Eleff S, Davis D, van Zijl PCM. Correlation of rapid changes in the average water diffusion constant and the concentrations of lactate and ATP breakdown products during global ischemia in cat brain. Magn Reson Med.. 1995;34:343-352.[Medline] [Order article via Infotrieve]
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Eleff SM, Maruki Y, Monsein LH, Traystman RJ, Bryan RN, Koehler RC. Sodium, ATP, and intracellular pH transients during reversible complete ischemia of dog cerebrum. Stroke.. 1991;22:233-241.
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