Sodium liquid ion exchanger microelectrode used to measure large extracellular sodium transients.

A new liquid membrane microelectrode has been developed that is easily fabricated and can measure fast sodium transients in the presence of potassium interference. It responds to a sudden change in sodium activity within 1 second. The electrode has been used to provide the first direct evidence of large sodium transients in the extracellular space of the brain of the catfish.

Astroglial acid-base dynamics in hyperglycemic and normoglycemic global ischemia.

Biochemical, histological, and physiological evidence suggest strongly that astrocytes may either defend or damage brain tissue, depending on the brain carbohydrate content preceding global ischemia (28,43). This paper will first review the concept of acidosis in ischemia and the possible role of severe, compartmentalized astrocytic acidosis in pan necrosis. Results are then presented demonstrating that astrocytes are also capable of maintaining an alkaline intracellular pH (pHi) during normoglycemic global ischemia. Mechanisms underlying depolarization-dependent astroglial alkalosis are then reviewed. Recent experiments indicate that bicarbonate (HCO3-) transport is a major mechanism by which astroglia not only alkalinize their interior but also acidify the interstitium. Maintenance of alkalosis during normoglycemic ischemia supports the hypothesis that astroglial HCO3- transport might ultimately protect neurons from excitotoxicity in ischemia without infarction (17). Inhibition of astroglial HCO3- transport may be a critical and requisite event, ultimately leading to compartmentalized astroglial acidosis and irreversible injury to all cell types.

Reactive astrocytosis from excitotoxic injury in hippocampal organ culture parallels that seen in vivo.

Reactive astrocytes influence not only the severity of brain injury, but also the capacity of brain to reshape itself with learning. Mechanisms responsible for astrogliosis remain unknown but might be best studied in vitro, where improved access and visualization permit application of modern molecular and cellular techniques. We have begun to explore whether gliosis might be studied in hippocampal organotypic cultures (HOTCs), where potential cell-to-cell interactions are preserved and the advantages of an in vitro preparation are still realized. Following HOTC exposure to N-methyl-D-aspartate (NMDA), dose-dependent changes occurred in the optical density (OD) values for the astrocytic immunohistochemical [immunostaining (IS)] markers glial fibrillary acidic protein (GFAP) and vimentin. Exposure of HOTCs to NMDA (10 microM) caused selective death in the CA1 hippocampal region and the dentate gyrus. It also significantly increased GFAP IS and vimentin IS OD values in these regions. Increased GFAP IS and vimentin IS OD values were also seen in CA3, a hippocampal region that displayed no cell death. Light microscopic examination revealed hypertrophied GFAP and vimentin IS cells, characteristic of reactive astrocytes. Cellular proliferation, as assessed by proliferating cell nuclear antigen IS, was also significantly increased in all three of these hippocampal regions. In contrast, exposure of HOTCs to a noninjurious level of NMDA (1 microM) caused only minor changes in GFAP IS and vimentin IS OD values but a significantly reduced cellular proliferation in all HOTC regions. These results show that reactive astrocytosis from excitotoxic injury of HOTC parallels changes seen in vivo after global ischemia. Furthermore, since resting astroglia within HOTCs are also similar to their counterparts in vivo, HOTCs may be used to examine mechanisms by which these cells are transformed into reactive species within tissue that resembles intact brain.

Neuronal nitric oxide synthase expression is induced in neocortical astrocytes after spreading depression.

Spreading depression (SD) confers either increased susceptibility to ischemic injury or a delayed protection. Because nitric oxide modulates ischemic injury, we investigated if altered expression of nitric oxide synthase (NOS) by SD could account for the effect of SD on ischemia. Furthermore, the identity of cells expressing NOS after SD is important, since SD results in heterogeneous, cell type-specific changes in intracellular environment, which can control NOS activity. Immunohistochemical, computer-based image analyses and Western blotting show that the number of neuronal NOS (nNOS)-positive cells in the somatosensory cortex was significantly increased at 6 hours and 3 days after SD (P < 0.05 and 0.01, respectively), whereas inducible NOS expression remained unchanged. Double-labeling of nNOS and glial fibrillary acidic protein identified these nNOS-positive cells as astrocytes. The effect of altered NO production on induced nNOS expression was examined by treating rats with sodium nitroprusside or NA-nitro-L-arginine methyl ester (LNAM) during SD. Increased nNOS expression was prevented by sodium nitroprusside and phenylephrine or phenylephrine alone, but not LNAM. Because SD increased astrocytic nNOS expression at time points correlating with both ischemic hypersensitivity and ischemic tolerance, the ability of SD to modulate ischemic injury must be complex, perhaps involving NOS but other factors as well.

Astrocytic acidosis in hyperglycemic and complete ischemia.

Nearly complete brain ischemia under normoglycemic conditions results in death of only selectively vulnerable neurons. With prior elevation of brain glucose, such injury is enhanced to include pancellular necrosis (i.e., infarction), perhaps because an associated, severe lactic acidosis preferentially injures astrocytes. However, no direct physiologic measurements exist to support this hypothesis. Therefore, we used microelectrodes to measure intracellular pH and passive electrical properties of cortical astrocytes as a first approach to characterizing the physiologic behavior of these cells during hyperglycemic and complete ischemia, conditions that produce infarction in reperfused brain. Anesthesized rats (n = 26) were made extremely hyperglycemic (blood glucose, 51.4 +/- 2.8 mM) so as to create potentially the most extreme acidic conditions possible; then ischemia was induced by cardiac arrest. Two loci more acidic than the interstitial space (6.17-6.20 pH) were found. The more acidic locus [4.30 +/- 0.19 (n = 5); range: 3.82-4.89] was occasionally seen at the onset of anoxic depolarization, 3-7 min after cardiac arrest. The less acidic locus [5.30 +/- 0.07 (n = 53); range 4.46-5.93)] was seen 5-46 min after cardiac arrest. A small negative change in DC potential [8 +/- 1 mV (n = 5); range -3 to -12 mV and 7 +/- 2 mV (n = 53); range +3 to -31 mV, respectively] was always seen upon impalement of acidic loci, suggesting cellular penetration. In a separate group of five animals, electrical characteristics of these cells were specifically measured (n = 17): membrane potential was -12 +/- 0.2 mV (range -3 to -24 mV), input resistance was 114 +/- 16 M omega (range 25-250 M omega), and time constant was 4.4 +/- 0.4 ms (range 3.0-7.9 ms). Injection of horseradish peroxidase into cells from either animal group uniformly stained degenerating astrocytes. These findings establish previously unrecognized properties of ischemic astrocytes that may be prerequisites for infarction from nearly complete ischemia: the capacity to develop profound cellular acidosis and a concomitant reduction in cell membrane ion permeability.

In vivo measurement of regional brain and tumor pH using [14C]dimethyloxazolidinedione and quantitative autoradiography. II: Characterization of the extracellular fluid compartment using pH-sensitive microelectrodes and [14C]sucrose.

We measured the extracellular (interstitial) pH (pHe) of RG-2 rat gliomas using H+-sensitive microelectrodes and estimated the volume of tumor extracellular space based on the tissue-plasma ratio of [14C]sucrose. The average RG-2 pHe was 7.63 +/- 0.15 (mean +/- SD, n = 6), whereas the average pHe of contralateral brain tissue was 7.34 +/- 0.10 (n = 3) and arterial pH was 7.36 +/- 0.02. RG-2 extracellular space water volume was estimated to be 0.3 ml water/g tissue. In separate experiments in normal, nontumored rats, intracellular pH (pHi) was calculated for nine gray and white matter regions based on measurements of tissue and plasma [14C]dimethyloxazolidinedione concentration. pHi values ranged from 6.80 to 6.94, and no consistent gray-white differences were observed. Our data suggest that tumor pHi is not more acidic than that of normal brain tissue and that the observed alkalinity of primary brain tumors is due to the presence of a large alkaline extracellular space.

Hydrogen ions kill brain at concentrations reached in ischemia.

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 microliter/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 less than or equal to 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.

Light and electron microscopic evaluation of hydrogen ion-induced brain necrosis.

Excessive accumulation of hydrogen ions in the brain may play a pivotal role in initiating the necrosis seen in infarction and following hyperglycemic augmentation of ischemic brain damage. To examine possible mechanisms involved in hydrogen ion-induced necrosis, sequential structural changes in rat brain were examined following intracortical injection of sodium lactate solution (pH 4.5), as compared with injections at pH 7.3. Following pH 7.3 injection, neuronal swelling developed between 1 and 6 h, but only a needle track wound surrounded by a thin rim of necrotic neurons and vacuolated neuropil was present 24 h after injection. In contrast, pH 4.5 injection produced neuronal necrosis as soon as 1 h after injection, followed by necrosis of astrocytes and intravascular thrombi at 3 and 6 h. Alterations common to both groups included vascular permeability to horseradish peroxidase, dilation of extracellular spaces, astrocyte swelling, capillary compression, and vascular stasis. These data suggest that neurons, astrocytes, and endothelia can be directly damaged by increased acid in the interstitial space. Lethal injury initially appeared to affect neurons, while subsequent astrocyte necrosis and vascular occlusion may damage tissue by secondary ischemia.

The effects of extracellular acidosis on neurons and glia in vitro.

Cerebral lactic acid, a product of ischemic anaerobic glycolysis, may directly contribute to ischemic brain damage in vivo. In this study we evaluated the effects of extracellular acid exposure on 7-day-old cultures of embryonic rat forebrain. Mixed neuronal and glial cultures were exposed to either lactic or hydrochloric acid to compare the toxicities of relatively permeable and impermeable acids. Neurons were relatively resistant to extra-cellular HCl acidosis, often surviving 10-min exposures to pH 3.8. In the same cultures, immunochemically defined astrocytes survived 10-min HCl exposures to a maximum acidity of pH 4.2. Similarly, axonal bundles defasciculated in HCl-titrated media below pH 4.4, although their constituent fibers often survived pH 3.8. Cell death occurred at higher pH in cultures subjected to lactic acidosis than in those exposed to HCl. Over half of forebrain neurons and glia subjected for 10 min to lactic acidification failed to survive exposure to pH 4.9. Longer 1-h lactic acid incubations resulted in cell death below pH 5.2. The potent cytotoxicity of lactic acid may be a direct result of the relatively rapid transfer of its neutral protonated form across cell membranes. This process would rapidly deplete intracellular buffer stores, resulting in unchecked cytosolic acidification. Neuronal and glial death from extracellular acidosis may therefore be a function of both the degree and the rapidity of intracellular acidification.

Eicosanoids and nitric oxide influence induction of reactive gliosis from spreading depression in microglia but not astrocytes.

Microglia and astrocytes are transformed into reactive glia (RG) by brain disease and normal function. Eicosanoids and nitric oxide (NO), two intercellular mediators, may influence gliosis. We investigated how drugs that alter production of these paracrine signals effect induction of glial reactivity from spreading depression. Unilateral (left) neocortical spreading depression was induced in 95 halothane anesthetized rats by intracortical injections of 0.5 M KCl, with or without drug treatment (five animals/group). Immunohistochemical staining (IS) intensity using the OX-42 and anti-glial fibrillary acidic protein (GFAP) antibodies determined reactivity in microglia and astrocytes, respectively. After 3 days, brains were processed for OX-42 and GFAP-IS and mean optical densities (OD) of IS were measured. Average OD's (for OX-42) and the log ratio (left/right) of OD's (OX-42 and GFAP) were compared to normal animals. Spreading depression induced significant log ratios for both OX-42- and GFAP-IS (P's < 0.01). However, dexamethasone (a glucocorticoid), nordihydroguaiaretic acid (a lipoxygenase inhibitor), and nitroprusside (a NO donor) prevented significant left sided and log ratio OD values for microglia (P's > 0.05). L-Name, a NO synthase inhibitor, caused significant increases in left and right OD's for microglia (P's < 0.05). Mepacrine, a phospholipase A2 inhibitor, Indomethacin, a cyclooxygenase inhibitor, and phenylephrine, an adrenergic agonist, did not prevent induction of significant OX-42 log ratios (P's < 0.01, 0.05, 0.01), and resulted in increases in left side OD's (P's < 0.01, 0.05, 0.05). Significant GFAP log ratios occurred after spreading depression in all drug groups, P's < 0.01. Thus, induction of reactivity in microglia is more sensitive to eicosanoids and NO than in astrocytes.

Characterization of inducible cyclooxygenase in rat brain.

Considerable debate exists regarding the cellular source of prostaglandins in the mammalian central nervous system (CNS). At least two forms of prostaglandin endoperoxide synthase, or cyclooxygenase (COX), the principal enzyme in the biosynthesis of these mediators, are known to exist. Both forms have been identified in the CNS, but only the distribution of COX 1 has been mapped in detail. In this study, we used Western blot analysis and immunohistochemistry to describe the biochemical characterization and anatomical distribution of the second, mitogen-inducible form of this enzyme, COX 2 in the rat brain. COX 2-like immunoreactive (COX 2-ir) staining occurred in dendrites and cell bodies of neurons, structures that are typically postsynaptic. It was noted in distinct portions of specific cortical laminae and subcortical nuclei. The distribution in the CNS was quite different from COX 1. COX 2-ir neurons were primarily observed in the cortex and allocortical structures, such as the hippocampal formation and amygdala. Within the amygdala, neurons were primarily observed in the caudal and posterior part of the deep and cortical nuclei. In the diencephalon, COX 2-ir cells were also observed in the paraventricular nucleus of the hypothalamus and in the nuclei of the anteroventral region surrounding the third ventricle, including the vascular organ of the lamina terminalis. COX 2-ir neurons were also observed in the subparafascicular nucleus, the medial zona incerta, and pretectal area. In the brainstem, COX 2-ir neurons were observed in the dorsal raphe nucleus, the nucleus of the brachium of the inferior colliculus, and in the region of the subcoeruleus. The distribution of COX 2-ir neurons in the CNS suggests that COX 2 may be involved in processing and integration of visceral and special sensory input and in elaboration of the autonomic, endocrine, and behavioral responses.

Long-term elevation of cyclooxygenase-2, but not lipoxygenase, in regions synaptically distant from spreading depression.

Eicosanoids, produced from arachidonic acid by cyclooxygenases (COXs) and lipoxygenases (LIPOXs), are involved in numerous brain processes. To explore if brief and noninjurious stimuli chronically alter expression of these enzymes, we examined the induction of COX-2 and LIPOX expression following unilateral neocortical spreading depression (SD). Expression was examined over time and in regions not experiencing SD (hippocampus) but synaptically connected to the site of stimulation (cortex). One hundred six male Wistar rats had SD induced via microinjection of 0.5 M KCl (0.5 M NaCl for sham) into left parietal cortex every 9 minutes for 1 or 3 hours. One hour before SD some animals received dexamethasone (Dex), mepacrine (Mep), indomethacin (Indo), nordihydroguaiaretic acid (Ndga), phenylephrine (Pe), sodium nitroprusside (Snp) with Pe, or N omega-nitro-L-arginine methyl ester (Lnam). Animals survived for 0, 3, or 6 hours, or 1, 2, 3, 7, 14, 21, or 28 days. Brains were processed immunohistochemically for COX-2 and LIPOX, and the optical density (OD) of the left and right cortex, dentate gyrus (DG), CA3, and CA1 immunoreactivity (IR) were measured. Induction was expressed as the log of left divided by right side OD for each region. COX-2 IR in the left cortex was elevated rapidly and was sustained for 21 days following SD. COX-2 IR was also elevated in the ipsilateral hippocampus not experiencing SD, with the rank order of induction as follows: DG > CA3 > CA1. Dex, Snp, and/or Pe significantly reduced the induction of COX-2. No changes in LIPOX IR were observed. These results show that long-term changes in COX-2 expression are induced by SD and these changes decrease with synaptic distance. Benign stimuli increase COX-2 expression and thus may influence brain function for extended periods and at distant locations.

Determination of extracellular bicarbonate and carbon dioxide concentrations in brain slices using carbonate and pH-selective microelectrodes.

The extracellular pH of the brain is subject to shifts during neural activity. To understand these pH changes, it is necessary to measure [H+], [HCO3-], [CO3(2-)] and [CO2]. In principle, this can be accomplished using CO3(2-) and pH-sensitive microelectrodes; however, interference from HCO3- and Cl-, and physiological changes in [HCO3-], complicate measurements with CO3(2-) electrodes. Calibration requires knowledge of slope response, interference constants and corrections for [HCO3-] shifts. We show that when [HCO3-] is altered at constant [CO2] in the absence of Cl-, the HCO3- interference cancels and the Nikolsky equation reduces to the Nernst equation for CO3(2-). Measurement of CO3(2-) slope response by this method yielded a value of 28.5 +/- 0.72 mV per decade change in [CO3(2-)]. In Cl(-)-containing solutions, interference coefficient for HCO3- and Cl- were determined by altering [HCO3-] at constant [CO2], changing [CO2] at constant [HCO3-], then solving the simultaneous Nikolsky equations for each transition. The mean interference constants corresponded to selectivity ratios of 245:1 and 1150:1 for CO3(2-) over HCO3- and Cl- respectively. To correct for possible changes in [HCO3-], the equilibrium relation between CO3(2-) and HCO3- was substituted into the Nikolsky equation to yield an equation in [CO3(2-)] and [H+]. By simultaneously measuring shifts in [H+] with a pH microelectrode, this equation is readily solved for [CO3(2-)]. These methods were tested by measuring [HCO3-] and [CO2] in experimental solutions, and in the extracellular fluid of rat hippocampal slices.

Optimization of multiplexed bead-based cytokine immunoassays for rat serum and brain tissue.

The ability to simultaneously quantify multiple signaling molecule protein levels from microscopic neural tissue samples would be of great benefit to deciphering how they affect brain function. This follows from evidence that indicates signaling molecules can be pleiotropic and can have complex interactive behavior that is regionally and cellularly heterogeneous. Multiplexed examination of tissue proteins has been exceedingly difficult because of the absence of available techniques. This void now has been removed by the commercial availability of bead-based immunoassays for targeted proteins that allow analyses of up to 100 (6-150 kDa) proteins from as little as 12 microl. Thus far used only for sera (human and mouse) and culture media, we demonstrate here that sensitive (as low as 2 pg/ml), wide-ranging (up to 2-32 000 pg/ml), accurate (8% intra-assay covariance) and reliable (4-7% inter-assay covariance) measurements can be made of nine exemplary cytokines (e.g., IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, GM-CSF, IFN-gamma, TNF-alpha) simultaneously not only from rat serum but, for the first time, also brain tissue. Furthermore, we describe animal handling procedures that minimize stress as determined by serum glucocorticoid levels since they can influence cytokine expression.

Focal elevations in neocortical interstitial K+ produced by stimulation of the fastigial nucleus in rat.

We studied whether K+, a potent cerebrovasodilator released by active neurons, participates in the increase in cortical cerebral blood flow (CBF) elicited by stimulation of the cerebellar fastigial nucleus (FN). Rats were anesthetized by continuous administration of halothane (1-3%), paralyzed and artificially ventilated. FN was stimulated electrically (8 s trains, 50 Hz, 5-10 V) through microelectrodes positioned stereotaxically. K+o (mM) was measured in sensory cortex by K(+)-sensitive micropipettes. In some experiments neocortical CBF was monitored continuously by laser-doppler flowmetry. Stimulation of the FN produced significant increases in K+o that averaged 0.91 +/- 0.16 mM (range 0.5-2.9 mM; n = 19) and were confined to sites corresponding to the intermediate cortical laminae (P less than 0.05, ANOVA). To determine whether such K+o elevations were able to produce increases in CBF comparable to those elicited by FN stimulation, cortical K+o was increased by superfusing the sensory cortex with 20-30 mM K+ in Ringer. K+o elevations of 2.8 +/- 0.6 mM increased CBF by 17 +/- 2% (n = 5), an increase considerably smaller than that elicited by FN stimulation in cerebral cortex. We conclude that K+ is unlikely to mediate the cortical cerebrovasodilation. Furthermore, the restricted spatial distribution of the K+o increase indicates that the cortical neural activity evoked by FN stimulation is highly focal. Thus the findings support the hypothesis that, in cortex, the vasodilation is mediated by activation of a restricted group of neural elements, perhaps neurons in laminae III-IV.

Acid-induced changes of brain protein buffering.

Excessive cellular acidosis is thought to enhance destruction of brain from ischemia. Protein denaturation may contribute to such injury although the behavior of brain proteins to acidosis is poorly defined. As a first approach to detect acid-induced changes in brain proteins and to characterize buffer content, homogenates were acidified for 20 min (as low as pH 3.1), returned to baseline pH (6.9), and then titrated. Titration curves show a significant (P less than 0.0001) and permanent increase in buffer content compared to controls when pH of acid exposure was 4.5-3.7 or less. Since acidity of pH 4.5 is rarely, if ever, achieved in vivo, protein denaturation from acidity alone is unlikely to account for necrosis of brain from ischemia.

Hydrogen ion buffering during complete brain ischemia.

As a first step to quantify [H+] changes in brain during ischemia we used H+-selective microelectrodes and enzyme fluorometric techniques to describe the relationship between interstitial [H+] ([H+]o) and peak tissue lactate after cardiac arrest. We found a step function relationship between [H+]o and tissue lactate rather than the linear titration expected in a homogeneous protein solution. Within a blood glucose range from 3-7 mM, brain lactate rose from 8-13 mmol/kg along with a rise in [H+]o of 99 +/- 6 nM(0.44 +/- 0.02 pH). At higher blood glucose levels (17-80 mM), brain lactate accumulated to levels of 16-31 mmol/kg; concurrently [H+]o rose by 608 +/- 16 nM (1.07 +/- 0.02 pH). The unchanging level of [H+]o between 8-13 and 16-31 mmol/kg lactate implies that [H+]o is at a steady-state, but not equilibrium with respect to [H+] in other brain compartments. We propose that ion-transport characteristics of astroglia account for the observed relationship of [H+]o to tissue lactate during complete ischemia and suggest that brain infarction develops after plasma membranes in brain cells can no longer transport ions to regulate [H+].

Acid-induced injury in elasmobranch brain.

Sodium lactate injection into rat brain produces coagulation necrosis consistent with infarction when pH0 is held at less than or equal to 5.30 for 20 min. Such injury may result from excessive astroglial acidification. If true, then brain damage from acidosis in elasmobranchs might evolve differently since glial reaction there to another necrotizing injury, exposure to extreme cold, is dissimilar from that seen in mammals. Accordingly, pH0 was monitored and sodium lactate (pH 4.00-7.00) injected into skate (Raja erinacea) cerebella. Necrosis was seen only when pH0 was less than or equal to 4.86 for 20 min; and pH0 rise after injections was unaffected by those which destroyed brain, and not slowed as in rat. Thus elasmobranchs are less susceptible to irreversible brain injury from acidosis, a capacity which may result from their lower body temperature compared to mammals.