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(Stroke. 1997;28:206-210.)
© 1997 American Heart Association, Inc.

Articles

Extracellular Potassium in a Neocortical Core Area After Transient Focal Ischemia

Gunilla Gido, MSc; Tibor Kristian, PhD Bo K. Siesjo, MD, PhD

the Laboratory for Experimental Brain Research, Experimental Research Centre, University Hospital, Lund, Sweden, and the Institute of Neurobiology, Slovak Academy of Sciences, Kosice, Slovak Republic.

Correspondence to Gunilla Gido, Laboratory for Experimental Brain Research, Wallenberg Neurocenter, University Hospital, S-221 85, Lund, Sweden. E-mail gunilla.gido@eforsk.lu.se.

	Abstract

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Background and Purpose Occlusion of the middle cerebral artery (MCAO) results in bioenergetic failure in the densely ischemic core areas. During reperfusion, transient recovery of the bioenergetic state is followed by secondary deterioration. In this study, we recorded the extracellular potassium concentrations in the cortical core during 2 hours of MCAO, as well as during recovery. One group of animals with recirculation periods of 6 to 8 hours was given the free radical spin trap -phenyl-N-tert-butyl nitrone (PBN).

Methods The experiments were performed on adult male Wistar rats (305 to 335 g). The right MCA was occluded by an intraluminal filament technique. For [K⁺]_e measurements a craniotomy was made over the right cortex, and an ion-sensitive microelectrode was lowered into the ischemic focus. Recording of [K⁺]_e was continued for 2 hours. After 48 hours of reperfusion, infarction size was estimated with 2,3,5-triphenyltetrazolium chloride.

Results During MCA occlusion, [K⁺]_e rose to 60 mmol/L. However, several animals showed transient (and partial) periods of repolarization accompanied by a decrease in [K⁺]_e. Immediately on reperfusion, the [K⁺]_e started to recover and reached baseline levels (2.5 mmol/L) within 3 to 5 minutes. During the first 6 hours of recovery, [K⁺]_e was stable at about 2.5 mmol/L, but after this period a moderate increase in the [K⁺]_e was observed. This was not observed in animals injected with PBN 1 hour after reperfusion.

Conclusions The data suggest that delayed cell membrane dysfunction, as reflected in a rise in [K⁺]_e, occurs after about 6 hours of reperfusion and that treatment with PBN in a single dose ameliorates or delays such deterioration of plasma membrane function.

Key Words: cerebral ischemia, focal • potassium • rats

	Introduction

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Focal ischemia such as that caused by MCAO leads to infarction, specifically destruction of both neurons and glial cells and eventually microvessels.^{1 2 3} Studies of reversible MCAO in rats have shown that the maximal size of the infarct is obtained when the vessel is occluded for 2 to 3 hours^{4 5} ; in primates, this time may be slightly longer.^{6 7} However, the infarct "matures" slowly over many hours, initially affecting neurons only.⁸ Studies of blood flow, bioenergetic state, and extracellular ion concentrations have defined a densely ischemic core ("focus") and a less densely ischemic perifocal zone (the "penumbra") in which cells are at risk, their fate being determined by the adequacy of the collateral circulation.^{2 9 10 11 12} Although the primary threat to cells in the penumbra is the underperfusion, it has been repeatedly emphasized that irregularly occurring depolarizations and ionic transients of the SD or "ischemic" type jeopardize the survival of neurons in the poorly perfused tissues.^{13 14 15 16 17} In fact, it has been assumed that glutamate antagonists ameliorate the ischemic lesions by reducing or arresting the depolarization waves.¹⁸

Recent results have demonstrated that recirculation after 2 hours of MCAO leads to an initial recovery of the bioenergetic state of core and penumbral tissues but that this is followed by secondary metabolic deterioration after 2 to 4 hours.¹⁹ Analyses of mitochondrial respirationin tissue homogenates suggest that, at least in part, this reflects secondary failure of mitochondrial function.²⁰ Furthermore, the spin trap PBN, which ameliorates focal ischemic damage after permanent or transient MCAO,^{21 22} was found to prevent the secondary deterioration of the bioenergetic state and mitochondrial function.^{19 23}

The present experiments were undertaken to assess how alterations in bioenergetic state and mitochondrial function are reflected in changes of DC potential and extracellular K⁺ concentration. To that end, [K⁺]_e and DC-potential shifts were measured during and after MCAO in a cortical area that usually becomes part of the final infarct. Three main questions were posed. First, when is recirculation followed by normalization of plasma membrane function of neurons and glia cells as reflected in [K⁺]_e? Second, after such normalization has occurred, are SD-like depolarizations observed spontaneously or after local applications of KCl? Third, when is a secondary rise in [K⁺]_e observed during recirculation, and how does this rise correlate to the secondary deterioration of bioenergetic state and mitochondrial function?

	Materials and Methods

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The experiments were performed on 45 adult male Wistar rats (305 to 335 g) of a specific pathogen-free strain (Møllegaard's Breeding Centre, Copenhagen, Denmark). Before the experiments, the animals were fasted overnight but allowed free access to tap water. All animals were anesthetized with 3.5% halothane and O₂-N₂O (30:70). Thereafter, most of them were allowed to spontaneously breathe into a nose mask delivering 1.5% halothane in O₂-N₂O. In these animals, a tail-artery catheter was inserted for blood pressure control, and an electrical temperature probe was inserted into the rectum to help keep the body temperature at 37°C during the MCAO. When the MCA had been occluded, the animals were allowed to wake up. One group of animals was injected with PBN (Sigma Chemical Co). A dose of 100 mg·kg^-1 of a solution containing 10 mg·mL^-1 was given after 1 hour of recirculation. The few animals that were not spontaneously breathing were intubated and connected to a respirator during the MCAO and were further handled as described below.

The right MCA was occluded with the intraluminal technique of Koizumi et al²⁴ as described previously.^{5 25} Briefly, a skin incision was made in the middle of the neck, and the right common carotid artery and the right external and internal carotid arteries were exposed. Thereafter, the external carotid artery was ligated, the internal carotid artery was encircled by a suture, and 0.1 mL heparin (150 IU·mL^-1) was given. A small incision was made in the common carotid artery 1 mm proximal to the carotid bifurcation, and the MCA occluder was inserted into the internal carotid artery and gently advanced 19 mm to the bifurcation. After the MCAO, the animals were allowed to wake up while freely moving in a box ventilated with cool air to prevent a rise in temperature.²⁵ In the animals kept on artificial ventilation, body temperature was kept at 37°C by adjustments of the external heating/cooling.

In three sham-operated animals in which the MCA was not occluded, the [K⁺]_e was recorded for 2 hours. These animals and many other control animals showed that [K⁺]_e is normally 3 mmol/L. The experimental animals were divided into six groups; [K⁺]_e was recorded during the 2 hours after MCAO or in the recirculation periods of 0 to 2, 2 to 4, 4 to 6, 6 to 8, or 19 to 21 hours. In one additional group, with recording after 6 to 8 hours, PBN was given after 1 hour of recirculation. All procedures followed the guidelines of the National Institutes of Health (Guide for Care and Use of Laboratory Animals) and were approved by the Lund University animal ethics committee. In all animals, catheters were inserted into the tail artery for blood gas analyses and recording of blood pressure and into a tail vein for infusion of muscle relaxant (Norcuron, Organ Teknika; 2 mg·mL^-1). The animals were placed in a head holder, and a craniotomy (OD, 2 mm) was made over the right cortex (coordinates: bregma anterior-posterior 0 mm, lateral 5.5 mm, and ventral 2.0 mm) for recording of [K⁺]_e in the core of the cortical lesion (Fig 1). To elicit an SD, an additional craniotomy was made over the frontal cortex, which is not affected by MCAO. SDs were elicited by application of 2 mol/L KCl to the exposed dura. The recording location was the same as described above. A temperature probe connected to an electrical heating pad was placed in the rectum. Because blood flow is close to normal in all reperfusion groups (R. Tsuchidate, Q.-P. He, M.-L. Smith, B.K. Siesjo, 1996), the head was not regularly temperature controlled. For the animals in which [K⁺]_e was recorded during occlusion, the heads were enclosed in a box to keep the brain temperature constant at 37°C.²⁶ After a small incision in the dura had been made, the electrodes were lowered into the parietal cortex by a motor-driven micromanipulator to a depth of 2.0 mm.

View larger version (36K): [in this window] [in a new window]   Figure 1. Typical infarcted area at the level of bregma. The black dot shows the recording position.

When the surgical procedures were completed, the halothane concentration in the gas mixture was decreased to 0.3% to 0.5%, and an infusion of the muscle relaxant Norcuron (1.2 mL·h^-1) was started. At this time, the recording of the extracellular potassium was started and continued for 2 hours. Physiological parameters were maintained within normal ranges during the experiment. After 2 hours of [K⁺]_e recording, the animals were allowed to regain consciousness and were disconnected from the respirator. They were then housed in cages with access to tap water and pellet food until decapitation for TTC staining (see below) to evaluate the area of infarction in the section where the electrode was placed.

The microelectrodes used were made from borosilicate glass tubing (OD, 1.5 mm; ID, 1.2 mm; Hilgenberg GmBH) and had a tip diameter of 5 to 6 µm.²⁷ They were double-barreled: one barrel recorded the [K⁺]_e and the other the DC-potential shift.^{28 29} The barrel for ion-activity recording was filled with 150 mmol/L KCl and a potassium ionophore (Cocktail A 60031, Fluka AG). The other barrel, which served as a reference, was filled with 150 mmol/L NaCl. Each barrel was connected to a high-input resistance amplifier with Ag-AgCl wires. The output was displayed and recorded on a Macintosh computer using the MacLab data-acquisition system. The animals were connected to the ground with a glass tube filled with 3% agar in 150 mmol/L NaCl placed subcutaneously in the neck. Calibrations of the potassium electrode were performed before and after the experiment in solutions containing 2, 10, 50, or 80 mmol/L KCl.

After 48 hours of recovery, the animals were anesthetized and killed by decapitation. Brains were quickly removed and chilled in ice-cold saline for 10 minutes. Coronal slices (1 mm) were cut in a tissue slicer, and the slices were immersed in a saline solution containing 1.0% TTC (Sigma Chemical Co) at 37°C for 20 minutes.³⁰ After the staining, the slice at the bregma level was manually depicted to verify the infarct size at the recording site. Animals with no neuronal necrosis at the recording site were discarded from the analysis.

The data presented were analyzed by ANOVA with post hoc Scheffe's test for comparison among several groups. In comparisons between two groups, Student's t test was used. All values are expressed as mean±SD.

	Results

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The physiological parameters are shown in the Table. In evaluating the changes in body temperature, it should be recalled that body temperature during operative interventions was deliberately kept at about 37°C, whereas spontaneously breathing animals maintain a temperature of about 38°C.³¹ The variation in blood glucose concentrations between 3.2 and 6.4 covers the physiological range.

View this table: [in this window] [in a new window]   Table 1. Physiological Parameters

In one group, recording of [K⁺]_e started about 15 minutes after MCAO and continued for 2 hours. In the other groups, recording was started at 2, 4, 6, or 19 hours of recirculation after 2 hours of MCAO and was continued for 2 hours. From the recordings, [K⁺]_e was calculated every second, with the obtained values being averaged to yield the mean [K⁺]_e for the 2-hour period.

During ischemia, [K⁺]_e either remained stable at values of about 60 mmol/L or showed a pattern of repeated depolarization waves. Such a pattern is illustrated in Fig 2. The maximal recorded level of [K⁺]_e was 58.0±12.3 mmol/L (mean±SD, n=11), and the total depolarization time was 79.2±17.5 minutes (range, 58 to 112 minutes). These depolarization times were associated with neuronal necrosis in all the animals included in the study; the size of the infarct is illustrated in Fig 1 (see above).

View larger version (10K): [in this window] [in a new window]   Figure 2. Representative graph demonstrating SD-like changes in [K+]e in the cortical ischemic focus during transient MCAO. Arrow indicates the onset of reperfusion.

Reperfusion invariably gave rise to a positive shift in the DC potential and to a rapid normalization of [K⁺]_e, which reached minimal values within 2 to 4 minutes (Fig 3). During the first 2-hour period after MCAO, [K⁺]_e stabilized at 2.51±0.73 mmol/L (n=12). This value is lower than that obtained in sham-operated animals, suggesting persistent activation of Na⁺/K⁺ transport.

View larger version (12K): [in this window] [in a new window]   Figure 3. The rapid shifts in [K+]e and the DC potential in the early reperfusion phase, with arrow indicating the onset of reperfusion.

Fig 4 demonstrates that [K⁺]_e remained at normal or subnormal values during the first 6 hours of recirculation. In the next 2-hour period (6 to 8 hours) [K⁺]_e rose, but this rise was moderate (5.18±2.19 mmol/L, n=8). Interestingly, no further rise was observed after 19 to 21 hours, ie, at the time when cell damage must have been relatively extensive. PBN, given in a dose of 100 mg·kg^-1 after 1 hour of recirculation, prevented the rise in [K⁺]_e concentration occurring at 6 to 8 hours after the start of reperfusion (see Fig 4).

View larger version (14K): [in this window] [in a new window]   Figure 4. Individual values showing the temporal profile of the [K+]e in the different recovery periods and in animals injected with PBN 1 hour after recirculation. Dashed line represents the normal [K+]e. *P<.05 or **P<.01 different from other groups by Scheffe's F test or *P<.05 difference with unpaired Student's t test when comparing the 6-to-8-hour groups without and with PBN.

During the first 6 hours of reperfusion, [K⁺]_e was remarkably stable. There were therefore no spontaneous depolarization events, nor was it possible to elicit an SD by local application of KCl at the dura on the frontal cortex. Clearly, the gradual deterioration of the bioenergetic state was not triggered (or preceded) by energy-consuming depolarization events.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
As extensively documented in the literature, ischemia of a certain density leads to bioenergetic failure and loss of ion homeostasis, with the latter involving a negative DC-potential shift, release of K⁺ from cells, and their uptake of Na⁺, Cl^-, and Ca²⁺. In addition, since uptake of Na⁺ and Cl^- occurs with osmotically obligated water, the ECF volume shrinks.^{32 33} It is therefore possible to use recordings of DC potential and [K⁺]_e to monitor plasma membrane function in energy-compromised tissues. There are two caveats, however. One is that [K⁺]_e is determined not only by translocation of K⁺ between intracellular fluids and ECFs but also by fluxes between blood and ECFs. In the short perspective (a few hours), the latter play a nonsignificant role because the blood-brain barrier permeability to K⁺ is low.³⁴ This may explain why [K⁺]_e during hypoglycemic coma rises to very high values, even though blood flow is normal or increased.^{35 36} However, during a 2-hour period of focal ischemia, when the K⁺ gradient between ECF and plasma is increased, there is a small loss of K⁺ from ischemic tissue.³⁷ With recirculation periods of 19 to 21 hours after 2 hours of ischemia, K⁺ transport across the blood-brain barrier cannot be neglected. Thus, one can envisage that cells lose K⁺ to ECFs and that the K⁺ gained by ECF is translocated from ECF to blood. Measurements of tissue K⁺ content are required to quantify this loss.

The other caveat is that documentation of a rise in [K⁺]_e gives no information on the source(s) of the K⁺ released. As discussed extensively in the literature, K⁺ released from neurons may be taken up by glial cells (see Reference 33). Thus, if K⁺ leaks out from a small number of neurons, it may be taken up by viable glial cells. The moderate rise in [K⁺]_e at 6 to 8 hours of recirculation probably reflects release of K⁺ that can no longer be buffered by glial cells; alternatively, glial function is compromised.

As discussed previously, MCAO of 2-hour duration is followed by a partial recovery of the bioenergetic state in the focus and penumbra after 1 hour of recirculation, with no further improvement after 2 hours, and secondary deterioration after 4 hours.¹⁹ Assessment of mitochondrial respiration activity in vitro yielded similar results (ie, an initial partial recovery) followed by secondary deterioration.^{20 23} However, the data on mitochondrial respiratory function showed a secondary deterioration already after 2 hours. This suggests that recirculation leads to an initial resynthesis of ATP, which is followed in turn by a gradual decline in mitochondrial respiratory functions and secondarily by deterioration of cellular bioenergetic state.

It is in this context that the present results fit in, since they give information on plasma membrane function in the critical period of reperfusion after 2 hours of focal ischemia. Our results demonstrate that even in an area destined to become part of the final infarct, reperfusion leads to complete normalization of [K⁺]_e, suggesting restoration of plasma membrane function. An interesting finding is that [K⁺]_e was not only normalized but also reduced to subnormal values. There are two possible reasons for this. One is that the Na-K pumps in the plasma membrane are upregulated. The other possibility is that the high level of [K⁺]_e during MCAO leads to increased efflux of K⁺ ions from ECF to plasma,³⁷ with an ensuing decrease in total tissue potassium content. This possibility is supported by data showing that the permeability surface product for Na⁺ transport across the blood-brain barrier increases during focal ischemia,³⁸ suggesting that K⁺ efflux may be enhanced. Regardless of the explanation, the results demonstrate that the recovery of mitochondrial function and ATP synthesis^{19 20 23} is accompanied by adequate resumption of Na⁺/K⁺ transport. The present results further demonstrate that a likely series of events during reperfusion encompasses reduction of mitochondrial respiratory capacity, deterioration of the bioenergetic state of the tissue, and plasma membrane dysfunction. This is also a logical series of events because failure of plasma membrane function must be preceded by bioenergetic failure, which in turn must be triggered by mitochondrial dysfunction.

Animals treated with PBN did not show a significant increase in [K⁺]_e after 6 to 8 hours of reperfusion, suggesting a preserved cellular energy production. Thus, the secondary perturbation of [K⁺]_e was prevented or delayed by postischemic administration of PBN. These data are in agreement with the finding that PBN prevents a deterioration of the bioenergetic state¹⁹ and of mitochondrial respiratory functions^{20 23} when given to animals during recirculation after transient focal ischemia. We realize the need to assess the effect of PBN after longer recovery periods, but we submit that such measurements should be combined with measurements of [Ca²⁺]_e and of total tissue electrolyte contents.

Although the results as discussed are relatively clear-cut, it remains to be explained why [K⁺]_e at 6 to 8 hours of recirculation does not increase above the 4- to 8-mmol/L level and why ongoing cell death after 19 to 21 hours of "recovery" is accompanied by a very moderate rise in [K⁺]_e. Additional experiments addressing this issue are clearly justified.

	Selected Abbreviations and Acronyms

 

ECF = extracellular fluid MCA(O) = middle cerebral artery (occlusion) PBN = -phenyl-N-tert-butyl nitrone SD = spreading depression TTC = 2,3,5-triphenyltetrazolium chloride

	Acknowledgments

 
This study was supported by the Swedish Medical Research Council (grant B96-14X-00263-32C), the US Public Health Service (grant 5R 01 NS 07838), and the Medical Faculty, University of Lund.

Received May 21, 1996; revision received August 19, 1996; accepted September 17, 1996.

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Editorial Comment

Raymond C. Koehler, PhD, Guest Editor

The Johns Hopkins University Department of Anesthesiology and Critical Care Medicine Baltimore, Md

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
With the early use of thrombolytic agents to provide reperfusion from embolic stroke, the question arises of whether neurons are capable of repolarization and restoration of cellular ionic gradients when ischemic duration is prolonged. Much of the experimental work on extracellular ionic measurements during reperfusion has focused on complete or near-complete global ischemia lasting less than 1 hour. However, ischemic duration is generally longer than 1 hour with the use of thrombolytic agents for embolic stroke. In the present study, the authors investigated the recovery of extracellular potassium activity after 2 hours of focal cerebral ischemia in rat neocortex. Remarkably, extracellular potassium activity rapidly recovered within a few minutes of reperfusion despite the 2 hours of severe ischemia. This result implies that most of the neurons and glia had functional mitochondria and intact plasma membranes, although direct proof would require intracellular potassium measurements.

By 6 to 8 hours of reperfusion, however, extracellular potassium activity began to increase to moderate levels. This observation implies that many neurons are beginning to die and release potassium and that astrocyte buffering of extracellular potassium becomes overwhelmed or dysfunctional. Thus, the therapeutic window can theoretically extend into reperfusion for several hours after a focal ischemic event. This hypothesis is supported by the authors' demonstration that administration at 1 hour of reperfusion of a nitrone-based free radical trap, which was previously found to reduce infarct volume, prevented the secondary increase in potassium activity at 6 to 8 hours. Therefore, the injury cascade is still in progress during reperfusion and can be therapeutically targeted at this time. These results support the design of therapies to be used in combination with thrombolytic agents at reperfusion.

	Selected Abbreviations and Acronyms

 

ECF = extracellular fluid MCA(O) = middle cerebral artery (occlusion) PBN = -phenyl-N-tert-butyl nitrone SD = spreading depression TTC = 2,3,5-triphenyltetrazolium chloride

Temp 1 indicates temperature during occlusion in freely moving rats; Temp 2, temperature in reperfusion period in freely moving rats; and Temp 3, temperature during ion recording. Values are mean±SD.

*P<.05 or P<.01 vs 0-2 h recovery, P<.05 vs 19-21 h recovery, and §P<.05 or ||P<.01 vs control with ANOVA followed by post hoc Scheffe's test.

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