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. Author manuscript; available in PMC: 2011 Feb 26.
Published in final edited form as: J Cereb Blood Flow Metab. 1987 Aug;7(4):379–386. doi: 10.1038/jcbfm.1987.80

Hydrogen Ions Kill Brain at Concentrations Reached in Ischemia

Richard P Kraig 1, Carol K Petito 2, Fred Plum 3, William A Pulsinelli 4
PMCID: PMC3045529  NIHMSID: NIHMS271401  PMID: 3611202

Summary

Elevation of brain glucose before the onset of nearly complete ischemia leads to increased lactic acid within brain. When excessive, such acidosis may be a necessary factor for converting selective neuronal loss to brain infarction from nearly complete ischemia. To examine the potential neurotoxicity of excessive lactic acid concentrations, we microinjected (0.5 µl/min) 150 mM sodium lactate solutions (adjusted to 6.50-4.00 pH) for 20 min into parietal cortex of anesthetized rats. Interstitial pH (pH0) was monitored with hydrogen ion–selective microelectrodes. Animals were allowed to recover for 24 h before injection zones were examined with the light microscope. Injectants produced brain necrosis in a histological pattern resembling ischemic infarction only when pH0 was ≤ 5.30. Nonlethal injections showed only needle tract injuries. Abrupt deterioration of brain acid-base homeostatic mechanisms correlated with necrosis since pH0 returned to baseline more slowly after lethal tissue injections than after nonlethal ones. The slowed return of pH0 to baseline after the severely acidic injections may reflect altered function of plasma membrane antiport systems for pH regulation and loss of brain hydrogen ion buffers.

Keywords: Glial cells, Hydrogen ions, Infarction, Ion-selective electrodes, Ischemia, Lactic acid

Infarction occurs when brain glucose is elevated above normal before the onset of nearly complete ischemia (Myers, 1979; Kalimo et al., 1981; Pulsinelli et al., 1982), circumstances known to raise neocortical lactate values during ischemia to 19 ≥ mmol/kg (Pulsinelii et al., 1982). Conversely, in neocortex only small- to medium-sized neurons are destroyed after nearly complete ischemia during normoglycemia (Pulsinelli and Brierley, 1979), which, in rat, is correlated with a neocortical lactate content of up to 13 mmol/kg (Pulsinelli and Duffy, 1983). These considerations have led to the postulate that infarction of brain from nearly complete ischemia is thought to result from excessive tissue lactacidosis (Plum, 1983).

Direct measurements of brain hydrogen ion concentration ([H+]) during ischemia have extended our understanding of the mechanisms by which acidosis can lead to infarction. Thorn and Heitmann (1954), using surface H+ -selective electrodes, first showed that brain becomes acidotic during ischemia. Twenty-seven years later Siemkowicz and Hansen (1981) documented with H+ -selective microelectrodes that acidic shifts occurred in interstitial pH (pH0) during nearly complete ischemia. Nemoto and Frinak (1981) also showed that a secondary acidosis occurs in interstitial space shortly after reperfusion from nearly complete ischemia. Kraig and colleagues (1985b) reported that this secondary acidosis can reach a pH as low as 5.4 under severely hyperglycemic conditions. More recently, we used pH0 and neocortical lactate content measurements to provide the first evidence that some cells become preferentially acidic during hyperglycemic and complete ischemia (Kraig et al., 1985a). Based on other data, obtained from simultaneous and direct measurements of pH0 and tissue carbon dioxide tension, we suggested that the cells that preferentially become more acidic than the remainder of ischemic brain are glia (Kraig et al, 1986a). Recently, direct measurements of intracellular pH (Kraig and Chesler, 1987; Kraig and Nicholson, 1987) have shown that glia become one to two orders of magnitude more acidic than the interstitial space during hyperglycemic and complete ischemia.

The present study reports that injection of sodium lactate solutions directly into mammalian brain produces coagulation necrosis when pH0 reaches or falls below 5.30 pH for 20 min. Preliminary reports of this work have appeared (Kraig et al., 1986b,c).

METHODS

Animal preparation and recording for acid injections

Male Wistar rats (250–400 g) were anesthetized with halothane and ventilated spontaneously with an oxygen-nitrogen mixture via an inhalation mask. Small craniotomies were made over parietal cortex 2 mm lateral to the sagittal suture and 2 mm posterior to the bregma. A tail artery was cannulated. Animals were then mounted in a stereotaxic head holder that was fitted with a water jacket to keep body temperature at 37°C. Warmed (37°C) normal Ringer solution (Kraig et al., 1983) flowed through a superfusion cup that surrounded the craniotomies. H+ -selective micro electrodes and a micro- (170 µm) injection needle (2–1361 fused silica needle from Supelco, Inc.) were mounted on a Narishige MT-5 micromanipulator so that the H+ -selective microelectrode was at a point immediately adjacent to the needle wall but 300 µm above the needle’s tip.

The assembled needle-H+-selective microelectrode array was advanced into the superfusion cup. First, normal Ringer solution equilibrated with 95% oxygen–5% carbon dioxide (7.35 pH at 25°C) flowed over the brain. Second, normal Ringer solution equilibrated with 100% carbon dioxide (6.11 pH at 25°C) replaced the first superfusate to establish a two-point in situ pH calibration. Finally, 7.35 pH Ringer solution again filled the superfusion cup and the array was advanced into the brain so that the needle tip reached 800 µm below the parietal pial surface. Arterial pH, arterial oxygen tension (PaO2), and arterial carbon dioxide tension (PaCO2) were stabilized and monitored with a Corning 158 blood gas analyzer. Blood glucose was measured with a Glucometer (Miles Laboratories, Inc.).

A constant infusion pump was used to inject sodium lactate (Fluka Chemical Co.) solutions. Sodium lactate solutions at 150 mM were adjusted to 4.00, 4.50, 5.00, 6.00, and 6.50 pH with concentrated hydrochloric acid and injected at a rate of 0.5 µ/min for 20 min. Recordings of pH0 were continued for 20–30 min after injections were completed. The needle-H+ -selective microelectrode was then withdrawn and the head incision sutured closed. Animals were allowed to recover with food and water provided ad lib. After 24 h animals were anesthetized, killed, and their brains processed for histological examination.

Histological techniques

Necrotic lesion diameters (Figs. 3 and 5) were determined from frozen sections (12 animals with an injection into each parietal cortex; n = 24). For frozen sectioning, animals were anesthetized with halothane, decapitated, and their brains removed and frozen in Freon over dry ice. Brains were stored at −80°C until processing, at which time 20-µm-thick histological sections were cut from frozen brains. Sections were stained with hematoxylin and eosin (Bancroft and Stevens, 1982). Necrotic lesion diameters were measured with a compound microscope that was calibrated with a stage micrometer. Areas of tissue necrosis stained less intensely with eosin and appeared pale with sharp borders demarcating the area of necrosis from normal brain.

FIG. 3.

FIG. 3

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Sodium lactate injection profile. Three double-barreled H+ -selective microelectrodes (left) were glued together and then positioned with an injection needle so that they were 300 µm above the needle’s tip and 0 (A), 200 (B), and 340 (C) µm away from the needle’s wall. Sodium lactate (150 mM at 4.00 pH) was injected at 0.5 µl/min for 20 min at a depth of 800 µm below the parietal pial surface. Twenty-micron-thick frozen sections stained with hematoxylin and eosin (right) showed the resultant necrotic lesion and the approximate H+-selective microelectrode recording points (black arrows). pH0 reached 4.11 (A) and 4.66 (B) at the two recording sites that were within the necrotic zone (left and center arrows), pH0 reached 7.19 (C) at the recording point (right arrow) that was outside the necrotic zone. Note that signals for B and C records were filtered through 10-Hz active filers while the signal for record A passed through a 200-Hz active filter. Thus, recording A shows more electrical noise (i.e., thicker line) than recording B or C.

FIG. 5.

FIG. 5

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Comparison of pH0 during injections to resultant size of necrotic lesions. Maximum pH0 values reached during injection of sodium lactate solutions are compared to the diameter of the resultant necrotic lesion measured from frozen sections (black dots). The latter were prepared and stained with hematoxylin and eosin (as in Fig. 3) after allowing animals to recover from acid injections for 24 h. Eight points below 100 µm in diameter (61 ± 5 µm SEM) represent diameter of the altered neuropil from needle tract damage and not necrosis. After these points, necrotic lesion diameter increased in a sigmoidal fashion compared to pH0. The line through these points is drawn by sight and emphasizes the observation that brain necrosis began when pH0 reached and then fell below ~5.30.

A second group of seven animals was processed by perfusion-fixation 24 h after acid injections to provide more detailed light microscopic analysis of the lesions. Animals were anesthetized with an intraperitoneal injection of urethane (1.6 g/kg; Sigma Chemical Co.) and then killed by intracardiac perfusion-fixation with a 1:1:8 (vol/vol/vol) solution of 37% formaldehyde, acetic acid, and methanol, respectively (FAM) (Pulsinelli and Brierley, 1979). Animals were decapitated after 10–15 min perfusion and heads stored at 4°C in FAM for 24 h. Brains were then removed, serially dehydrated, and embedded in paraffin. Seven-micron serial sections were stained with hematoxylin and eosin or luxol fast blue and cresyl violet. Histological characterization of lesions was done by one of the authors (C.P.) who was blinded to the pH of the injectants.

H+ -selective microelectrode construction and calibration

H+ -selective microelectrodes based on tridodecylamine (Ammann et al., 1981) were constructed, calibrated, and used as previously described (Kraig et al., 1983, 1985a, 1986a).

RESULTS

Preinjection physiologic variables

Blood physiologic variables were consistent with those of other halothane-anesthetized and spontaneously ventilating animals (Kraig et al., 1985a,b, 1986a; Kraig and Cooper, 1987) (Table 1). Animals were slightly hypercapnic probably because of a halothane-induced reduction in brainstem ventilatory drive stimuli (Smith, 1971). In addition, haiothane was the most likely cause for the mild reduction seen in arterial blood pressure (Deutsch, 1971). Anesthetic levels of halothane (0.75–1.5%) were associated with an absence of withdrawal to hind-limb pin prick and an average PaCO2 of 70 mm Hg and an average systolic arterial blood pressure of 111 mm Hg. Otherwise, animals were normoxic and normothermic and had normal hematocrits. Animals were mildly hyperglycemic (glucose 7.7 ± 0.2 mM) presumably because they were not fasted.

TABLE 1.

Physiologic variables

Brain pH0 7.42 ± 0.12
Arterial pH 7.30 ± 0.01
PaCO2 (mm Hg) 70 ± 1
PaO2 (mm Hg) 119 ± 3
Arterial glucose (mM) 7.7 ± 0.2
Hematocrit (%) 44 ± 0.0
Systolic blood pressure (mm Hg) 111 ± 1
Rectal temperature (°C) 37.2 ± 0.1
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Values are means ± SEM, n = 38.

Brain pH0 was more alkaline (Fig. 1; Table 1) than has previously been described. Brain pH0 is normally 0.05–0.10 pH more acid than arterial pH in halothane-anesthetized and spontaneously ventilating rats (Kraig et al., 1985a, 1986a; Kraig and Cooper, 1987). However, in experiments reported here, pH0 was 0.12 pH on average more alkaline (7.42 ± 0.12 pH) than arterial blood (7.30 ± 0.01 pH). The cause of this alkalosis is unknown. If bicarbonate (HCO3−) remains the sole H+ buffer of the interstitial space, then the interstitial alkalosis of 7.42 could result from an increase in interstitial HCO3 − concentration or a fall in tissue carbon dioxide tension. Changes in interstitial HCO3− concentration are unlikely to account for the observed alkalosis since membrane transport processes generally require minutes of activity to influence concentration changes on either side of a membrane (Roos and Boron, 1981) and the alkalosis was present as soon as the injection needle–H+ -selective microelectrode array entered the brain interstitial space. Instead, a reduction of brain tissue carbon dioxide tension of 20 mm Hg is a more likely cause for the rise in pH0 from 7.25–7.35 that is usually seen (Kraig et al., 1983, 1985a, 1986a; Kraig and Cooper, 1987) to 7.42 that is reported here (Table 1). Tissue carbon dioxide tension might have been lowered near the H+ -selective microelectrode recording tip by an increased diffusion distance from the micro electrode tip to brain that was created by the relatively large injection cannula.

FIG. 1.

FIG. 1

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pH0 changes associated with insertion of injection needle and H+-selective microelectrode. Penetration of the microinjection cannula–H+-selective microelectrode array into brain [seen as equivalent and parallel vertical deflections in direct current (DC) (bottom) and pH0 (top) records] often induced a spreading depression. DC and pH0 changes immediately after array penetration into brain were typical of spreading depression (Kraig et al., 1983): (a) slow negative DC signal of ~15 mV, which, after almost 5 min, was associated with the return of spontaneous electrical activity (seen as increased thickness of DC line) to involved parietal cortex; (b) alkaline- then acid-going transient in pH0 that reached an acidic peak of ~7.00 pH. After spreading depression pH0 returned to a baseline of 7.42 ± 0.12 pH (n = 37). Under normal circumstances brain is slightly (0.05–0.10 pH) more acid than blood (7.30 ± 0.01 pH). Why baseline pH0 was often more alkaline in these experiments is unknown but probably reflects an effect associated with the blunt trauma from penetration of the large (170 µm) injection needle into brain. For example, if bicarbonate remained the sole H+ buffer of the interstitial space, then a reduction of ~ 20 mm Hg in tissue carbon dioxide tension at the H+-selective microelectrode recording site would account for the rise in pH0 from 7.25–7.35 usually seen to 7.42 reported here. Such a reduction in tissue carbon dioxide tension might result from the increased diffusion distance from normal brain to the H+-selective microelectrode recording area created by the injection cannula.

Penetration of the injection needle-H+ -selective micro electrode array into parietal cortex often resulted in a single wave of spreading depression (Fig. 1), a gross, transient, perturbation in ion and metabolite homeostasis of involved nervous tissue (Bureš et al., 1974). A single spreading depression or even up to 40 repetitive spreading depressions in neocortex does not result in detectable injury to brain when involved areas are examined by light microscopic techniques 24 h later (R. P. Kraig, unpublished observations). Thus, it is unlikely that spreading depression associated with sodium lactate injections caused any irreversible injury to brain.

pH0 changes associated with sodium lactate injections

Microinjection of sodium lactate solutions quickly resulted in a steady-state pH0 (Table 2; Fig. 2) that reflected the varying influence of brain physicochemical H+ buffers. pH0 plateaued 0.24–0.25 pH higher than injectants during introduction of the less acidic (6.50 and 6.00 pH) solutions. The difference between plateau pH0 and injectant pH increased to 0.46 and 0.66 during introduction of 5.00 and 4.50 pH solutions, respectively (Table 2). Such disparity between injectant pH and pH0 is likely to reflect an increase in brain physicochemical H+ buffer content from acid-induced protein denaturation. This conclusion stems from the observation (Kraig and Wagner, 1987) that the H+ buffer content of brain proteins can be increased by exposure to acid. Brain proteins, like other selected proteins (Hashemeyer and Hashemeyer, 1973), contain H+ buffer groups that are normally available for titration and others that are sequestered from aqueous media. With exposure to acid, such proteins begin to lose their normal three-dimensional structure and thus expose more H+ buffer moieties to the aqueous environment. Maximal exposure of sequestered H+ buffer groups in brain homogenates occurs at ~4.5 pH (Kraig and Wagner, 1987). Similar acid-induced denaturation of brain proteins may also occur in vivo, since maximal H+ buffering of injectants was seen at 4.5 pH (Table 2). Reduction of brain H+ buffer content through acid-induced protein precipitation may account for the observation that plateau pH0 was only 0.18 higher than the injectant during introduction of 4.00 pH sodium lactate.

TABLE 2.

Maximum pH0 from acid injections

Injectant pH pH0
4.00 4.18 ± 0.06 (n = 10)
4.50 5.16 ± 0.14 (n = 9)
5.00 5.46 ± 0.14 (n = 7)
6.00 6.24 ± 0.11 (n = 6)
6.50 6.75 ± 0.12 (n = 4)
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Values are means ± SEM.

FIG. 2.

FIG. 2

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Composite of pH0 changes from sodium lactate injections. Typical recordings are shown of pH0 changes from intracerebral injections of 150 mM sodium lactate solutions adjusted to 4.00 (a), 4.50 (b), 5.00 (c), 6.00 (d), and 6.50 (e) pH. pH0 was recorded with double-barreled H+-selective microelectrodes positioned adjacent to but 300 µm behind the tip of a microinjection needle. The latter in turn was positioned 800 µm beneath the parietal pial surface. Constant infusion (0.5 µl/min for 20 min) began at the upward arrow and stopped at the downgoing arrow. pH0 fell to an approximately constant level (see Table 2) within 1–2 min after injections began. pH0 returned to baseline after injections. Solutions (6.50 and 6.00 pH) that did not result in brain necrosis (e and d) were correlated with pH0 records that returned to baseline at analogous and rapid rates after injection of acid stopped. Similarly, 5.00 pH sodium lactate (c) was correlated with a pH0 record that returned to baseline at the same rate as after injection of solutions at 6.00 and 6.50 pH. Thus, although injectant pH was 5.00 and brain was destroyed in a volume with an approximate diameter of the injection needle, pH0 reached 5.55 only at the H+ -selective microelectrode recording site in viable brain tissue and beyond the edge of necrosis. On the other hand, sodium lactate injectants at 4.50 and 4.00 pH (b and a) that caused pH0 to fall below 5.30 pH (see Fig. 3) produced a necrotic zone that included the H+-selective microelectrode recording point. After these latter two injections, pH0 returned to baseline more slowly. This suggests that H+ homeostatic mechanisms are impaired immediately after exposure to H+, which ultimately results in brain necrosis.

The rate of return of pH0 to baseline after microinjections correlated with the ultimate effect of injectants on brain. Injectants (6.50, 6.00, 5.00 pH) that failed to produce brain necrosis at the site of the H+ -selective microelectrode recording resulted in pH0 changes that returned to baseline at rapid and virtually identical rates. Alternatively, pH0 returned to baseline more slowly after injection of solutions (4.50 and 4.00 pH) that produced brain necrosis. These results suggest that H+ -related homeostatic mechanisms are impaired immediately after exposure to solutions sufficiently acidic to produce brain necrosis (see Discussion).

Histological examination of injection zones

Injection of sodium lactate solutions at a rate of 0.5 µl/min caused a new steady-state pH0 to be reached that extended ~300 µm to either side of the midline of the injection cannula (Fig. 3, left). The size of the involved volume of brain provided an easily observable area of necrosis with the more acidic injectants (Fig. 3, right). Necrotic diameters of acidic lesions were measured from frozen sections (i.e., Fig. 3, right) and cellular detail was analyzed from perfusion-fixed brains stained with hematoxylin and eosin (Fig. 4). Sodium lactate injections that resulted in pH0 values at or below 5.30 pH (Fig. 5) were associated with a well-circumscribed zone of coagulation necrosis. The necrotic zone was surrounded by tissue where neurons showed eosinophilic necrosis, astrocytes had enlarged nuclei, and monocytes were present. Injections producing brain pH0 above 5.30 pH were associated only with damage from the injection needle–H+ -selective microelectrode array insertion into brain and alteration of the neuropil from fluid injections. Such injections revealed a central zone of necrosis (61 ± 5 µm in diameter) that resulted from needle tract damage that was surrounded by an altered neuropil extending 100–300 µm to either side of the injection tract, which was vacuolated and contained occasional neurons showing eosinophilic necrosis (Petito et al., 1987).

FIG. 4.

FIG. 4

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Necrotic lesions from sodium lactate injections. Luxol fast blue–and cresyl violet–stained section is shown. Necrotic lesion was produced by injection (0.5 µl/min for 20 min) of 150 mM sodium lactate (4.50 pH) at a depth of 800 µm below the parietal pial surface. pH0 was recorded as in Fig. 2 and reached 4.85 during the injection. A well-circumscribed area of coagulation necrosis was produced. Most neurons within this area appeared as eosinophilic ghosts or showed changes similar to those seen in ischemia with shrunken eosinophilic cytoplasm and pyknotic nuclei. Nuclear fragmentation was evident and nuclear debris was scattered throughout the lesion. Arrow (left) points to an eosinophilic neuronal ghost, which is also shown at a higher magnification (right). The area of necrosis was surrounded by a thin band of altered neuropil. Neurons with eosinophilic necrosis were present in this band. In addition, perineuronal vacuolization, proliferation of microglial cells, swollen astrocytic nuclei, and a mild proliferation of capillaries were evident in this border zone. Calibration bar in lower right-hand corner of each figure is 100 µm in length. (Modified from Kraig et al., 1986b.)

DISCUSSION

This investigation shows that injection of lactic acid into brain sufficient to produce a pH0 at or below 5.30 for a duration of 20 min produced local areas of tissue necrosis. This level of acidity closely approximates that proposed to occur in glia (i. e., 5.2 pH) during nearly complete ischemia (Kraig et al., 1986a) and is less than that directly observed with intracellular H+ -selective microelectrodes to occur within these cells during complete ischemia under hyperglycemic conditions (Kraig and Chesler, 1987; Kraig and Nicholson, 1987). Furthermore, such hyperglycemia is associated with a pH0 in brain of 5.4 pH shortly after reperfusion from nearly complete ischemia (Kraig et al., 1985b). Thus, 5.2–5.4 pH is a level of acidity that can occur in brain during ischemia and is shown here to cause brain necrosis. The pattern of necrosis observed here from sodium lactate injections resembled that seen in ischemic brain infarction: A zone of coagulation necrosis is surrounded by a rim of necrotic neurons and reactive glial cells.

In these experiments, the first hint of irreversible injury to brain consisted of a slower than normal rate for pH0 to return to baseline after sodium lactate injections (Fig. 2). Adjustment of pH0 toward baseline after acid injections can be proposed to operate by at least two mechanisms. First, passive diffusion or bulk flow of excess acid away from or H+ buffers toward the injection zones could raise pH0 toward baseline. After nonlethal injections, such movement of proton equivalents would be restricted to the interstitial space because of retained plasma membrane integrity. Alternatively, if cell membranes were immediately damaged during severely acidic injections, proton equivalents would be expected to diffuse into injured cells from the interstitial space. Movement of proton equivalents across leaky (and irreversibly injured) cell membranes probably occurred after injection of the most acidic (i.e., 4.00 pH) sodium lactate since this level of acidity has previously been shown to permanently denature brain proteins (Kraig and Wagner, 1987) and has been shown here to be associated with exhaustion of brain H+ buffer capacity. Other lethal injectants (i.e., 4.50 and 5.00 pH) may not have immediately destroyed cell membranes since they were associated with the largest degree of brain H+ buffering (i. e., greatest disparity between injectant pH and plateau pH0 during injections). After the introduction of nonlethal 4.50 and 5.00 pH injectants, pH0 could have returned toward baseline by a second mechanism: exchange transport (antiport) of proton equivalents across plasma membranes. For example, HCO3−, the principal H+ buffer of interstitial space, is thought to emanate from glia by antiport with interstitial chloride (Kimelberg and Bourke, 1982; Kraig et al., 1986a). After nonlethal injections an increased rate of HCO3− delivery to the interstitial space might hasten the return of pH0 to baseline. Similarly, if cell membranes remain intact, HCO3− might be the only H+ buffer added to the interstitial space during and after the introduction of lethal injectants at 4.50 and 5.00 pH. The intermediately slowed return of pH0 to baseline seen with these latter injectants (Fig. 2) may reflect a pH sensitivity for HCO3− delivery to interstitial space via Cl−/HCO3− antiport. In red blood cells, Cl−/HCO3− antiport is a pH-dependent process that is almost completely inhibited at 5.0–5.5 pH (Cabantchik et al., 1978). If Cl−/HCO3− antiport in glia has a similar pH sensitivity, then the slowed return of pH0 after introduction of 4.50 and 5.00 pH injectants could indicate an acid-induced inhibition of HCO3− secretion by glia.

The cellular mechanisms by which acid injections produce brain necrosis are unknown but seem likely to accompany excessive cellular acidosis. When pH0 is lowered to ≤ 5 pH, one can expect the intracellular milieu of neurons and glia to become progressively acidotic because of pH-related effects on plasma membrane antiport systems. Barnacle muscle, for example, becomes progressively acidified when pH0 is lowered below 6.0 pH (Boron et al., 1979), presumably by reversal of Na+/H+ antiport, so that interstitial protons enter the cell instead of being expelled from cells in the normal manner. A similar mechanism coupled to acid-induced inhibition of Cl− /HCO3− antiport could acidify mammalian neurons and glia during sufficiently acidic sodium lactate injections. Both Na +/H+ and Cl−/HCO3− antiport are present in mammalian astrocytes (Kimelberg and Bourke, 1982) but they have not been demonstrated in mammalian neurons. Their documentation, however, in several other animal cell types (Roos and Boron, 1981; Thomas, 1984) including vertebrate neurons (Chesler and Nicholson, 1985) implies that Na +/H+ and Cl−/HCO3− antiporters probably exist in mammalian neurons as well.

The molecular basis by which excessive cellular acidity destroys brain is unknown. Preliminary experiments by Pulsinelli and Petito (1983) using an acid injection paradigm suggest that the acidic reduction of ferric to ferrous iron and release of ferrous iron from organic stores in the tissue may catalyze the generation of active oxygen radicals with subsequent lipid peroxidation of cell membranes (Pulsinelli et al., 1985).1

Acknowledgment

This study was supported by the National Institutes of Neurological and Communicative Disorders and Stroke grant nos. NS-19108, NS-003346, BRSG S07, RR05396 and a Teacher Investigator Development Award (no. NS-00767) as well as a Redel Foundation and Du Pont Foundation grant to R.P.K. The expert technical assistance of D. Vecchio and C. Pike is gratefully acknowledged.

Abbreviation used

FAM

1: 1:8 (vol/vol/vol) solution of 37% formaldehyde, acetic acid, and methanol, respectively

Footnotes

1

Sodium lactate solutions were originally injected into brain through stainless-steel needles (Pulsinelli and Petito, 1983). These early injections produced brain necrosis when injectant pH was ~6.0. When the injection paradigm was changed to one that used fused silica instead of stainless steel, the threshold for brain necrosis dropped to 4.50–5.00 for injectant pH (Table 2) and 5.30 for pH0 (Fig. 5). Acidity in the range of 6.50-6.00 pH enhances the solubility of ferrous iron so that more acidic sodium lactate solutions that passed through stainless-steel needles contained higher quantities of iron than less acidic solutions (w. A. Pulsinelli, unpublished observations). Therefore, the additional injection of iron in the early experiments may have increased intracerebral ferrous iron; that in tum could have catalyzed the formation of active oxygen radicals with subsequent lipid peroxidation of cellular membranes (Pulsinelli et al., 1985). Because of this possibility, all solutions used in the present experiments were never exposed to iron-containing materials.

Contributor Information

Richard P. Kraig, Department of Neurology, Cornell University Medical College, New York, New York, U.S.A.

Carol K. Petito, Department of Pathology (Neuropathology), Cornell University Medical College, New York, New York, U.S.A.

Fred Plum, Department of Neurology, Cornell University Medical College, New York, New York, U.S.A..

William A. Pulsinelli, Department of Neurology, Cornell University Medical College, New York, New York, U.S.A.

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