ARTICLE, CELLULAR/MOLECULAR

Neuroprotective Effect of High Glucose Against NMDA, Free Radical, and Oxygen–Glucose Deprivation through Enhanced Mitochondrial Potentials

So Y. Seo, Eun Y. Kim, Harriet Kim and Byoung J. Gwag
Journal of Neuroscience 15 October 1999, 19 (20) 8849-8855; https://doi.org/10.1523/JNEUROSCI.19-20-08849.1999

Abstract

Cultured cortical neurons maintained in 25 mm glucose underwent a widespread neuronal death after exposure to NMDA, AMPA, and kainate. Among these, NMDA toxicity was substantially reduced in neurons maintained in 100 mm glucose. NMDA-induced increase in [Ca2+]i and reactive oxygen species was attenuated in neurons maintained in high glucose that revealed increased mitochondrial membrane and redox potentials as determined using rhodamine 123 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.p-trifluoromethoxy-phenylhydrazone, KCN, and rotenone, the selective inhibitors of mitochondrial potential, abrogated neuroprotective effect of high glucose against NMDA. The neuroprotective action of high glucose was extended against oxygen or combined oxygen–glucose deprivation. The present study provides evidence that prolonged exposure of cortical cells to high glucose attenuates NMDA- and free radical-mediated neuronal death via enhanced mitochondrial function.

Glucose in brain supplies energy essential for maintenance of the nervous system. Deficiency in glucose that results from hypoglycemia or ischemic insults can trigger neuronal injuries. Balance in ion homeostasis is disturbed, which in turn results in membrane depolarization and massive release of neurotransmitters, including glutamate (Siesjo, 1988; Erecinska and Silver, 1989). The extracellular accumulation of glutamate results in neuronal death by activating ionotropic glutamate receptors sensitive to NMDA or AMPA–kainate (Choi, 1988). In addition, neurons impaired of energy metabolism appear to be highly sensitive to excitotoxicity (Simon et al., 1984; Wieloch, 1985; Monyer et al., 1989; Cebers et al., 1998). Intracellular free Ca2+ and reactive oxygen species (ROS) have been well documented as causative mediators of excitotoxicity (Choi, 1988; Coyle and Puttfarcken, 1993). In addition, several studies suggest that brain supplied with excess glucose becomes more vulnerable to ischemic injuries, presumably by increasing lactic acid production and generation of ROS (Smith et al., 1986; Nedergaard, 1987; Lundgren et al., 1992; Li et al., 1998). However, administration of high glucose before hypoxic–ischemia has been reported to reduce brain damage (Ginsberg et al., 1987; Zasslow et al., 1989; Kraft et al., 1990; Vannucci et al., 1996). In addition to this beneficial effect of hyperglycemia, maneuvers increasing glucose entry into neurons were shown to protect neurons from glutamate neurotoxicity (Ho et al., 1995), stroke or seizure (Lawrence et al., 1995, 1996), and mitochondrial toxins (Dash et al., 1996). Addition of glucose metabolites, pyruvate and malate, attenuated neuronal death after exposure to glutamate or H2O2 (Desagher et al., 1997; Ruiz et al., 1998). Recognizing the apparent controversial effects of high glucose on neuronal injuries, we set out experiments to examine how prolonged exposure to high glucose modulates excitotoxicity, oxidative stress, and deprivation of oxygen or glucose induced in primary cortical cell cultures.

MATERIALS AND METHODS

Primary cortical cell culture. Neocortices were prepared from brains of fetal ICR mice at 14–15 d gestation and mechanically triturated as previously described (Noh and Gwag, 1997). Dissociated cells were plated on 24 well plates (five hemispheres per plate, ∼105 cells per well) in a plating medium consisting of Eagle’s minimal essential media (MEM; Earle’s salts, 11090–081) supplemented with 5% horse serum, 5% fetal bovine serum, and 2 mm glutamine. Two different groups of cultures were prepared by supplementing plating medium with 21 mmglucose (final glucose concentration, 25 mm) or 96 mm glucose (final glucose concentration, 100 mm). Under both conditions, glia become confluent at 7–8 d in vitro (DIV). Thereafter, overgrowth of glial cells was halted by 2 d exposure to 10 μm cytosine arabinoside. Cultures were then fed twice a week with plating medium lacking fetal serum. Cultures were maintained at 37°C in a humidified 5% CO2 atmosphere.

Neurotoxicity experiments. Cortical cell cultures (DIV 12–14) were washed in MEM (bicarbonate-free, 11700–010) supplemented with 26.6 mm bicarbonate and 21 mm glucose. Cultures were then exposed to excitotoxins (NMDA, AMPA, or kainate) or free radical-inducing agents [Fe2+ or buthionine-S,R-sulfoximine (BSO)] for 24 hr.

For oxygen or glucose deprivation, cortical cell cultures (DIV 15–17) were transferred to an anaerobic chamber containing 5% CO2, 10% H2, and 85% N2 as described before (Gwag et al., 1997). For oxygen deprivation, culture medium was replaced with deoxygenated balanced salt solution containing (in mm): 143.6 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, 5.5 glucose, and 10 mg/l phenol red. For combined oxygen and glucose deprivation, culture medium was replaced with deoxygenated balanced salt solution lacking glucose. To terminate injuries, cultures were taken out of the anaerobic chamber, added with concentrated glucose to the exposure solutions (final glucose concentration, 5.5 mm), and placed to the aerobic CO2 incubator.

Analysis of neuronal death. Overall cell injury was assessed microscopically under phase-contrast optics or by measuring amount of lactate dehydrogenase (LDH) released into the bathing medium 24 hr after neurotoxic insults as previously described (Koh and Choi, 1987). The percent neuronal death was normalized to the mean LDH value released 24 hr after continuous exposure to 500 μm NMDA (= 100) or a sham control (= 0).

Calcium imaging. Measurement of intracellular free calcium concentration ([Ca2+]i) was performed using a Ca2+-sensitive indicator fura-2 under a fluorescence microphotometry (Grynkiewicz et al., 1985). Cortical cell cultures (DIV 12) grown on a glass-bottom dish were loaded with 5 μm fura-2 AM plus 2% Pluronic F-127 for 30 min at room temperature. Cells were washed three times with a salt solution containing (in mm): 120 NaCl, 5 KCl, 2.3 CaCl2, 15 glucose, 20 HEPES, and 10 NaOH, pH 7.4. The fura-2 fluorescent signals (Ex = 340/380 nm; Em = 510 nm) were acquired with a Nikon Diaphot inverted microscope and CCD camera. Fura-2 ratio images were analyzed using a Quanticell 700 system (Applied Imaging).

ROS imaging. Cortical cell cultures (DIV 12) grown on a glass-bottom dish were loaded with 5 μmdichlorodihydrofluorescein diacetate (DCDHF-DA; Molecular Probes, Eugene, OR) plus 2% Pluronic F-127 in HEPES-buffered control salt solution (HCSS) containing (in mm): 120 NaCl, 5 KCl, 1.6 MgCl2, 2.3 CaCl2, 15 glucose, 20 HEPES, and 10 NaOH. Cultures were incubated for 20 min at 37°C, washed three times with HCSS, and the fluorescence signal of DCF (Ex = 490 nm; Em = 510 nm), the oxidation product of DCDHF-DA by free radicals, was analyzed on the stage of a Nikon Diaphot inverted microscope equipped with a 100 W Xenon lamp. To minimize background signal caused by direct oxidation of DCDHF-DA by illumination at 490 nm, intracellular levels of ROS were analyzed within 3 sec after illumination using a Quanticell 700 system (Applied Imaging).

Measurement of mitochondrial transmembrane potential.Cortical cell cultures (DIV 12) grown on a glass-bottom dish were loaded with 5 μm rhodamine 123 (Molecular Probes, Eugene, OR), a fluorescent dye indicating mitochondrial transmembrane potential (Bellomo et al., 1991), in HEPES-buffered control salt solution (HCSS) containing (in mm): 120 NaCl, 5 KCl, 1.6 MgCl2, 2.3 CaCl2, 15 glucose, 20 HEPES, and 10 NaOH. Cultures were incubated for 10 min at 37°C, washed three times with HCSS, and the fluorescence signal (Ex = 480 nm; Em = 520 nm) of rhodamine 123 was analyzed on the stage of a Nikon Diaphot inverted microscope equipped with a 100 W Xenon lamp. Background fluorescence signal of rhodamine 123 was determined on a glass-bottom dish without cells and subtracted from rhodamine 123 signals obtained in cortical neurons maintained in 25 and 100 mm glucose. All images were analyzed using a Quanticell 700 system (Applied Imaging).

MTT assay. The mitochondrial dehydrogenase activity that cleaves 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was used to determine mitochondrial redox potential in a quantitative colorimetric assay (Mosmann, 1983). Cortical cell cultures (DIV 12) were incubated with 100 μg/ml MTT in PBS for 1 hr at 37°C. The supernatant was then aspirated, and the formazan product was dissolved in dimethylsulfoxide and analyzed at 570 nm.

RESULTS

Attenuation of NMDA-induced neurotoxicity in cortical cell cultures maintained in high glucose

We first examined the possibility that excitotoxicity would be altered in cortical cell cultures grown in high glucose. Cortical cell cultures (DIV 12) maintained in 25 mm glucose showed swelling of neuronal cell body within 6 hr after exposure to 20 μm NMDA (Fig.1A). The NMDA-induced cell swelling was not produced in cortical cell cultures maintained in 100 mm glucose. However, cortical neurons (DIV 12) maintained in 25 mm or 100 mm glucose underwent cell body swelling after exposure to 40 μm kainate or 10 μm AMPA (data not shown). Whereas cortical cell cultures grown in 25 mm glucose showed dose-dependent neuronal death 24 hr after exposure to 10–40 μm NMDA, 3–30 μm AMPA, or 20–80 μm kainate, cultures grown in 100 mm glucose were highly resistant to neuronal death induced by NMDA, but not the other excitotoxins (Fig.1B).

Fig. 1.
Fig. 1.

Excitotoxicity in cortical cell cultures maintained in 25 or 100 mm glucose. A,Phase-contrast photomicrographs of cortical cell cultures (DIV 12) grown in 25 or 100 mm glucose after 6 hr exposure to a sham wash or 20 μm NMDA. Note degenerating neurons evident by cell body swelling (white arrows). Scale bar, 30 μm.B, Cortical cultures (DIV 12–14) grown in 25 or 100 mm glucose were exposed to 10–40 μm NMDA, 3–30 μm AMPA, or 20–80 μm kainate for 24 hr. Neuronal death was assessed by measuring LDH efflux into the bathing medium, mean ± SEM (n = 12 culture wells per each condition), scaled to the mean LDH value released after 24 hr exposure to 500 μm NMDA (=10). *Significant difference from relevant control group (cultures grown in 25 mm glucose) at p < 0.05 using ANOVA and Student–Neuman–Keuls test.

Fura-2 fluorescence microphotometry was performed to determine if cortical neurons grown in high glucose would override rise in [Ca2+]i after exposure to NMDA. In cortical neurons grown in 25 mmglucose, [Ca2+]iwas increased gradually over 120 min after exposure to 20 μm NMDA. Although the baseline levels of [Ca2+]i were not different between cortical neurons maintained in 25 and 100 mm glucose, NMDA-induced rise in [Ca2+]i was markedly reduced in neurons grown in 100 mm glucose (Fig.2). Accumulation of ROS after activation of NMDA receptors has been implicated as a necessary route to neuronal death (Dyken, 1994; Dugan et al., 1995). In cortical neurons grown in 25 mm glucose, addition of 20 μm NMDA increased levels of ROS within the 3 hr that was analyzed using DCDHF-DA, a redox-sensitive dye (Fig. 3). The baseline levels of [ROS]i were similar in cortical neurons maintained in 25 and 100 mm glucose, but [ROS]i produced after activation of NMDA receptors was significantly reduced in cortical cell cultures grown in 100 mm glucose.

Fig. 2.
Fig. 2.

Prolonged exposure to high glucose reduces accumulation of neuronal [Ca2+]i after exposure to NMDA. In cortical cell cultures (DIV 12–14), [Ca2+]i was analyzed using fura-2 at indicated times after addition of 20 μm NMDA, mean ± SEM (n = 30–35 neurons randomly chosen from four culture wells for each condition), scaled to mean neuronal [Ca2+]i after a sham control (= 100). *Significant difference from relevant control group (cultures grown in 25 mm glucose) at p < 0.05 using ANOVA and Student–Neuman–Keuls test.

Fig. 3.
Fig. 3.

Prolonged exposure to high glucose reduces generation of [ROS]i after exposure to NMDA. In cortical cell cultures (DIV 12–14), [ROS]i in cortical neurons at indicated times after exposure to 20 μm NMDA was analyzed by measuring fluorescence intensity of oxidized DCDHF-DA, mean ± SEM (n = 25 neurons randomly chosen from three culture wells for each condition), scaled to mean neuronal [ROS]i after a sham control (= 100). *Significant difference from relevant control group (cultures grown in 25 mm glucose) at p < 0.05 using ANOVA and Student–Neuman–Keuls test.

The neuroprotective effect of high glucose requires enhanced mitochondrial potential

Activation of NMDA receptor results in selective uptake of Ca2+ into mitochondria and subsequent production of ROS that appears to mediate neuronal death (Dugan et al., 1995; Reynolds and Hastings, 1995; Peng and Greenamyre, 1998). Glucose entered into cells likely enhances mitochondrial potentials that play a central role in regulation of [Ca2+]i and ROS. This possibility was examined using the positively charged and lipophilic rhodamine 123 that permeates into the negatively charged mitochondria and therefore reflects the mitochondrial transmembrane potential (Johnson et al., 1980; Emaus et al., 1986). The fluorescence signal of rhodamine 123 was significantly increased in cortical neurons grown in 100 mm glucose compared to 25 mmglucose (Figs. 4A,B). We also analyzed mitochondrial redox potential by measuring reduction of the tetrazolium salt MTT by α-nicotinamide adenine dinucleotide (NADH) dehydrogenase. Prolonged exposure to high glucose enhanced mitochondrial redox potential as shown by increased MTT reduction (Fig. 4C).

Fig. 4.
Fig. 4.

Prolonged exposure to high glucose increases mitochondrial membrane and redox potentials in cortical neurons.A, Fluorescence photomicrographs (stained with rhodamine 123) of cortical neurons (DIV 12) grown in 25 or 100 mmglucose. Scale bar, 30 μm. B, Mitochondrial membrane potential was analyzed by measuring the fluorescence intensity of rhodamine 123 in cortical neurons (DIV 12–14) grown in 25 or 100 mm glucose, mean ± SEM (n = 40 neurons randomly chosen from three culture wells for each condition).C, Mitochondrial redox potential was analyzed by measuring reduction of MTT at 570 nm in cortical neurons (DIV 12–14) grown in 25 or 100 mm glucose, mean ± SEM (n = 12 neurons randomly chosen from three culture wells for each condition). *Significant difference between values from cultures in 25 and 100 mm glucose at p< 0.05 using independent-samples t test.

Additional experiments were performed to determine if the enhanced mitochondrial potentials by high glucose would be required for attenuation of NMDA neurotoxicity. Inclusion of 0.1 μmp-trifluoromethoxy-phenylhydrazone (FCCP), a hydrophobic protonophore reducing mitochondrial membrane potential, was not toxic itself but abrogated the neuroprotective action of high glucose against NMDA (Fig. 5). The neuroprotective effect of high glucose against NMDA was also reversed with inclusion of subtoxic doses of rotenone or KCN that reduces mitochondrial redox potential by blocking NADH dehydrogenase or cytochrome oxidase. We performed additional experiments to examine if effect of high glucose reducing NMDA-induced accumulation of [Ca2+]i and [ROS]i would be abrogated in the presence of FCCP, KCN, or rotenone. In cortical neurons maintained in 100 mm glucose, neither [Ca2+]i nor [ROS]i was accumulated after exposure to 100 μm KCN, 0.1 μm FCCP, or 0.1 μm rotenone for 4–12 hr (data not shown). However, concurrent treatment with 100 μm KCN, 0.1 μm FCCP, or 0.1 μmrotenone elevated accumulation of [Ca2+]i and [ROS]i after exposure to 20 μm NMDA (Table1). These results imply that prolonged exposure of cortical neurons to high glucose attenuates NMDA-induced accumulation of [Ca2+]i and [ROS]I through enhanced mitochondrial potentials.

Fig. 5.
Fig. 5.

The neuroprotective effects of high glucose are reversed by inhibitors of mitochondrial potentials. A,Cortical cultures (DIV 12–14) grown in 100 mm glucose were exposed to 20 μm NMDA, alone or in the presence of 0.1 μm FCCP, 0.1 μm rotenone (RO), or 100 μm KCN. Sister cultures were exposed to the same doses of FCCP, RO, or KCN alone. Neuronal death was assessed 24 hr later by LDH assay as described above, mean ± SEM (n = 12 culture wells per each condition). *Significant difference from relevant control at p < 0.05 using ANOVA and Student–Neuman–Keuls test.

View this table:
Table 1.

Inhibitors of mitochondrial potentials abrograte effect of high glucose reducing NMDA-induced accumulation of [Ca2+]i and [ROS]i

Attenuation of free radical neurotoxicity in cortical cell cultures maintained in high glucose

We next examined the neuroprotective potential of high glucose against oxidative stress. Cortical cell cultures grown in 25 mm glucose underwent widespread neuronal death 24 hr after exposure to 50 μm Fe2+ or 1 mm BSO that produces ROS by a Fenton reaction or depletion of glutathione, respectively. This free radical-mediated neurotoxicity was substantially decreased in cortical cell cultures grown in 100 mm glucose (Fig. 6). Accumulation of ROS after exposure to Fe2+was significantly reduced in cortical neurons grown in 100 mm glucose compared to cortical neurons grown in 25 mm glucose (Fig. 7). The baseline levels of [ROS]i were similar between cortical cells maintained in 25 and 100 mm glucose.

Fig. 6.
Fig. 6.

High glucose protects cortical neurons against oxidative stress. Cortical cell cultures (DIV 12–14) grown in 25 or 100 mm glucose were exposed to 50 μmFeCl2 or 10 mm BSO for 24 hr. Neuronal death was analyzed by LDH assay as described above, mean ± SEM (n = 12 culture wells per each condition). *Significant difference between LDH values from cultures in 25 and 100 mm glucose at p < 0.05 using independent-samples t test.

Fig. 7.
Fig. 7.

Decreased production of [ROS]i in cortical neurons grown in high glucose. [ROS]i in cortical neurons were analyzed at indicated times after exposure to 50 μm Fe2+ by measuring fluorescence intensity of oxidized DCDHF-DA, mean ± SEM (n= 25 neurons randomly chosen from three culture wells for each condition). *Significant difference from relevant control group (cultures grown in 25 mm glucose) at p< 0.05 using ANOVA and Student–Neuman–Keuls test.

Cortical neurons maintained in high glucose are protected from deprivation of oxygen or combined oxygen and glucose

Cortical cell cultures grown in 25 mm glucose produced swelling of neuronal cell body at 6 hr and subsequent neuronal death at 24 hr after deprivation of oxygen for 6–14 hr (Fig.8A,B). Maintaining cultures in 100 mm glucose markedly protected cortical neurons from the oxygen deprivation for 6–12 hr. Cortical neurons maintained in high glucose were also resistant to insults induced by deprivation of oxygen and glucose for 60–100 min (Fig. 8C).

Fig. 8.
Fig. 8.

Cortical cell cultures grown in high glucose are resistant to neurotoxicity after deprivation of oxygen or combined oxygen–glucose. A, Phase-contrast photomicrographs of cortical neurons (DIV 16) grown in 25 or 100 mm glucose taken immediately after 8 hr deprivation of oxygen. Note widespread degeneration of cortical neurons maintained in 25, not 100 mm glucose (arrows). Scale bar, 30 μm.B,C, Cortical cell cultures (DIV 14–16) grown in 25 or 100 mm glucose were deprived of oxygen for 0, 2, 4, 6, 8, 10, 12, or 14 hr (B). Cortical cultures (DIV 14–16) were deprived of oxygen and glucose for 0, 20, 40, 60, 80, 100, or 120 min (C). Neuronal death was analyzed 24 hr later by measuring LDH as described, mean ± SEM (n = 12 culture wells per each condition). *Significant difference from relevant control group (cultures grown in 25 mm glucose) at p < 0.05 ANOVA and Student–Neuman–Keuls test.

DISCUSSION

We have demonstrated that cortical neurons maintained in high glucose are spared against excitotoxicity induced by NMDA, but neither AMPA nor kainate. Neurons maintained in high glucose reveal enhanced mitochondrial transmembrane and redox potential that likely counteract toxic accumulation of [Ca2+]i and [ROS]i. The neuroprotective effects of high glucose are extended to injuries by oxygen or glucose deprivation as well as oxidative stress.

Activation of NMDA or AMPA–kainate glutamate receptors results in rapid influx of ions such as Na+, Ca2+, and Cl− that in turn causes cell body swelling and lysis (Choi, 1988). In the present study, prolonged exposure to high glucose attenuated NMDA neurotoxicity without influencing AMPA- and kainate-induced neuronal death. Recognizing the unique property of the NMDA receptor complex as a major route of Ca2+ entry, the preferential neuroprotective effect of high glucose against NMDA may be attributable to reducing toxic actions of Ca2+. In support of this, administration of excess glucose to PC12 cells was reported to reduce the depolarization-induced increase of [Ca2+]i (Chan and Greenberg, 1991). In the present study, NMDA-induced [Ca2+]iaccumulation was not observed in cortical neurons maintained in high glucose. Moreover, NMDA-induced production of [ROS]i, which results from Ca2+-dependent uncoupling of mitochondrial electron transport (Malis and Bonventre, 1986; Lafon-Cazal et al., 1993; Kiedrowski and Costa, 1995; Reynolds and Hastings, 1995), was reduced in cortical neurons maintained in high glucose. The present findings that cortical neurons maintained in high glucose are resistant against neurotoxicity by NMDA, but neither kainate nor AMPA, provide further evidence supporting causative role of Ca2+ and ROS for NMDA-mediated excitotoxicity (Tymianski et al., 1993; Reynolds and Hastings, 1995;Dugan et al., 1995; Stout et al., 1998).

Accumulated [Ca2+]i is extruded or can be redistributed through ATP-dependent reuptake into the endoplasmic reticulum and passive entry into the mitochondria matrix (Wang and Thayer, 1996; Peng et al., 1998). Mitochondrial transmembrane potential was markedly increased in cortical neurons maintained in high glucose. This increased potential can drive ATP production that enhances efflux of Ca2+and therefore counteracts NMDA-induced [Ca2+]iaccumulation. Alternatively, the mitochondrial membrane potential enhanced by high glucose can detoxify excess accumulation of [Ca2+]i by accelerating entry of Ca2+ to intracellular Ca2+ pools irrespective of ATP production. In support of this, hippocampal and cortical neurons treated with 2.5 mm pyruvate and 1.5 mmmalate show enhanced Ca2+ entry into the endoplasmic reticulum or the mitochondria without increasing ATP production (Villalba et al., 1994). With enhanced mitochondrial redox potential by high glucose that results in substantial reduction of [ROS]i, NMDA-induced accumulation of [ROS]i as well as [Ca2+]i can be lowered to subtoxic levels in cortical neurons. Selective inhibitors of mitochondrial membrane and redox potential reversed the neuroprotective effect of high glucose against NMDA, suggesting that the enhanced mitochondrial potentials are necessary for the neuroprotective effect of high glucose.

The mitochondria play a role in generation and processing of [ROS]i. Abnormal changes in mitochondrial DNA and proteins have been observed in brain during aging and neurodegenerative process (Coyle and Puttfarcken, 1993; Beal, 1995). The mitochondrial dysfunction can result in accumulation of [ROS]i and cause neuronal death. Mitochondrial toxins such as N-methyl,4-phenyl-1,2,3,6 tetrahydropyridine and 3-nitropropionic acid appear to produce ROS-mediated neurotoxicity via inhibition of electron transporters (Beal et al., 1993; Smith and Bennett, 1997). In the present study, increasing mitochondrial potentials protected neurons against Fe2+and BSO by interfering with accumulation of [ROS]i. Further study will be needed to determine whether the neuroprotective effect of increased mitochondrial potentials can be extended to other physiological and pathological cell deaths mediated through ROS.

NMDA neurotoxicity and oxidative stress have been well documented as mechanisms underlying hypoxic–ischemic brain injuries. The protective effects of high glucose that attenuated NMDA- and ROS-mediated neuronal death were also observed against deprivation of oxygen or glucose. The neonatal rat administrated with high glucose is resistant to hypoxic–ischemic injuries (Palmer and Vannucci, 1993). In contrast to the beneficial effects of high glucose, systemic administration of glucose before ischemia accentuates brain damage in the adult animals after hypoxic–ischemic injuries (Sieber and Traystman, 1992). Increased accumulation of lactic acid and [ROS]i has been correlated with the worsening effects of glucose. However, neuroprotective effect of high glucose after prolonged exposure does not conflict with the deleterious one of glucose systemically administered 1 hr before onset of hypoxic–ischemic injury. In our study, acute treatment (1–3 d) with high glucose did not protect cortical neurons from exposure to NMDA or oxygen–glucose deprivation (S. Seo and B. Gwag, unpublished data).

Prolonged exposure to high glucose protects selectively against NMDA- and ROS- dependent neurotoxicity. The enhanced mitochondrial membrane and redox potentials by high glucose demonstrate as key mechanisms underlying the neuroprotective effects of high glucose. Maneuvers increasing mitochondrial potentials can be merited to treat acute and chronic neurodegenerative diseases that depend upon excess activation of NMDA receptors or toxic accumulation of [ROS]i.

Footnotes

  • This work was supported by Korea Science and Engineering Foundation through the Brain Disease Research Center at Ajou University (B.J.G.).

    Correspondence should be addressed to Dr. Byoung J. Gwag, Department of Pharmacology, Ajou University School of Medicine, San 5, Wonchon-dong, Paldal-gu, Suwon, Kyungki-do 442–749, Korea.

REFERENCES

  1. ↵
    1. Beal MF
    (1995) Aging, energy, and oxidative stress in neurodegenerative diseases. Ann Neurol 38:357–366.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Beal MF,
    2. Brouillet E,
    3. Jenkins BG,
    4. Ferrante RJ,
    5. Kowall NW,
    6. Miller JM,
    7. Storey E,
    8. Srivastava R,
    9. Rosen BR,
    10. Hyman BT
    (1993) Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid. J Neurosci 13:4181–4192.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Bellomo G,
    2. Fulceri R,
    3. Albano E,
    4. Gamberucci A,
    5. Pompella A,
    6. Parola M,
    7. Benedetti A
    (1991) Ca(2+)-dependent and independent mitochondrial damage in hepatocellular injury. Cell Calcium 12:335–341.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Cebers G,
    2. Cebere A,
    3. Liljequist S
    (1998) Metabolic inhibition potentiates AMPA-induced Ca2+ fluxes and neurotoxicity in rat cerebellar granule cells. Brain Res 779:194–204.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Chan J,
    2. Greenberg DA
    (1991) Effects of glucose on calcium channels in neural cells. Neurosci Lett 121:34–36.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Choi DW
    (1988) Glutamate neurotoxicity and diseases of the nervous system. Neuron 1:623–634.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Coyle JT,
    2. Puttfarcken P
    (1993) Oxidative stress, glutamate, and neurodegenerative disorders. Science 262:689–695.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Dash R,
    2. Lawrence M,
    3. Ho D,
    4. Sapolsky R
    (1996) A herpes simplex virus vector overexpressing the glucose transporter gene protects the rat dentate gyrus from an antimetabolite toxin. Exp Neurol 137:43–48.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Desagher S,
    2. Glowinski J,
    3. Premont J
    (1997) Pyruvate protects neurons against hydrogen peroxide-induced toxicity. J Neurosci 17:9060–9067.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Dugan LL,
    2. Sensi SL,
    3. Canzoniero LM,
    4. Handran SD,
    5. Rothman SM,
    6. Lin S,
    7. Goldberg MP,
    8. Choi DW
    (1995) Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-d-aspartate. J Neurosci 15:6377–6388.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Dyken JA
    (1994) Isolated cerebral and cerebellar mitochondria produce free radicals when exposed to elevated Ca2+ and Na2+: implication for neurodegeneration. J Neurochem 63:584–591.
    OpenUrlPubMed
  12. ↵
    1. Emaus RK,
    2. Grunwald R,
    3. Lemasters JJ
    (1986) Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim Biophys Acta 23:436–448.
    OpenUrl
  13. ↵
    1. Erecinska M,
    2. Silver IA
    (1989) ATP and brain function. J Cereb Blood Flow Metab 9:2–19.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Ginsberg MD,
    2. Prado R,
    3. Dietrich WD,
    4. Busto R,
    5. Watson BD
    (1987) Hyperglycemia reduces the extent of cerebral infarction in rats. Stroke 18:570–574.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Grynkiewicz G,
    2. Poenie M,
    3. Tsien R
    (1985) A new generation of Ca2+-indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Gwag BJ,
    2. Koh JY,
    3. DeMaro JA,
    4. Ying HS,
    5. Jacquin M,
    6. Choi DW
    (1997) Slowly triggered excitotoxicity occurs by necrosis in cortical cultures. Neuroscience 77:393–401.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Ho DY,
    2. Saydam TC,
    3. Fink SL,
    4. Lawrence MS,
    5. Sapolsky RM
    (1995) Defective herpes simplex virus vectors expressing the rat brain glucose transporter protect cultured neurons from necrotic insults. J Neurochem 65:842–850.
    OpenUrlPubMed
  18. ↵
    1. Johnson LV,
    2. Walsh ML,
    3. Chen LB
    (1980) Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci USA 77:990–994.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Kiedrowski L,
    2. Costa E
    (1995) Glutamate-induced destabilization of intracellular calcium concentration homeostasis in cultured cerebellar granule cells: role of mitochondria in calcium buffering. Mol Pharmacol 47:140–147.
    OpenUrlAbstract
  20. ↵
    1. Koh JY,
    2. Choi DW
    (1987) Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Methods 20:83–90.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Kraft SA,
    2. Larson CP Jr.,
    3. Shuer LM,
    4. Steinberg GK,
    5. Benson GV,
    6. Pearl RG
    (1990) Effect of hyperglycemia on neuronal changes in a rabbit model of focal cerebral ischemia. Stroke 21:447–450.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Lafon-Cazal M,
    2. Pietri S,
    3. Culcasi M,
    4. Bockaert J
    (1993) NMDA-dependent superoxide production and neurotoxicity. Nature 364:535–537.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Lawrence MS,
    2. Ho DY,
    3. Dash R,
    4. Sapolsky RM
    (1995) Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss. Proc Natl Acad Sci USA 92:7247–7251.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Lawrence MS,
    2. Sun GH,
    3. Kunis DM,
    4. Saydam TC,
    5. Dash R,
    6. Ho DY,
    7. Sapolsky RM,
    8. Steinberg GK
    (1996) Overexpression of the glucose transporter gene with a herpes simplex viral vector protects striatal neurons against stroke. J Cereb Blood Flow Metab 16:181–185.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Li PA,
    2. Gisselsson L,
    3. Keuker J,
    4. Vogel J,
    5. Smith ML,
    6. Kuschinsky W,
    7. Siesjo BK
    (1998) Hyperglycemia-exaggerated ischemic brain damage following 30 min of middle cerebral artery occlusion is not due to capillary obstruction. Brain Res 804:36–44.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Lundgren J,
    2. Smith ML,
    3. Siesjo BK
    (1992) Effects of dimethylthiourea on ischemic brain damage in hyperglycemic rats. J Neurol Sci 113:187–197.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Malis CD,
    2. Bonventre JV
    (1986) Mechanism of calcium potentiation of oxygen free radical injury to renal mitochondria: a model for post-ischemic and toxic mitochondrial damage. J Biol Chem 261:14201–14208.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Monyer H,
    2. Goldberg MP,
    3. Choi DW
    (1989) Glucose deprivation neuronal injury in cortical culture. Brain Res 483:347–354.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Mosmann TR
    (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Nedergaard M
    (1987) Transient focal ischemia in hyperglycemic rats is associated with increased cerebral infarction. Brain Res 408:79–85.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Noh JS,
    2. Gwag BJ
    (1997) Attenuation of oxidative neuronal necrosis by a dopamine D1 agonist in mouse cortical cell cultures. Exp Neurol 146:604–608.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Palmer C,
    2. Vannucci RC
    (1993) Potential new therapies for perinatal cerebral hypoxia-ischemia. Clin Perinatol 20:411–432.
    OpenUrlPubMed
  33. ↵
    1. Peng TI,
    2. Greenamyre JT
    (1998) Privileged access to mitochondria of calcium influx through N-methyl-d-aspartate receptors. Mol Pharmacol 53:974–80.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Peng TI,
    2. Jou MJ,
    3. Sheu SS,
    4. Greenamyre JT
    (1998) Visualization of NMDA receptor-induced mitochondrial calcium accumulation in striatal neurons. Exp Neurol 149:1–12.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Reynolds IJ,
    2. Hastings TG
    (1995) Glutamate induced the production of reactive oxygen species in cultured forebrain neurons following NMDA receptor application. J Neurosci 14:3318–3327.
    OpenUrl
  36. ↵
    1. Ruiz F,
    2. Alvarez G,
    3. Pereira R,
    4. Hernandez M,
    5. Villalba M,
    6. Cruz F,
    7. Cerdan S,
    8. Bogonez E,
    9. Satrustegui J
    (1998) Protection by pyruvate and malate against glutamate-mediated neurotoxicity. NeuroReport 9:1277–1282.
    OpenUrlPubMed
  37. ↵
    1. Sieber FE,
    2. Traystman RJ
    (1992) Special issues: glucose and the brain. Crit Care Med 20:104–114.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Siesjo BK
    (1988) Hypoglycemia, brain metabolism, and brain damage. Diabetes Metab Rev 4:113–144.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Simon RP,
    2. Swan JH,
    3. Griffiths T,
    4. Meldrum BS
    (1984) Blockade of N-methyl-d-aspartate receptors may protect against ischemic damage in the brain. Science 226:850–852.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Smith ML,
    2. von Hanwehr R,
    3. Siesjo BK
    (1986) Changes in extra- and intracellular pH in the brain during and following ischemia in hyperglycemic and in moderately hypoglycemic rats. J Cereb Blood Flow Metab 6:574–583.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Smith TS,
    2. Bennett JP
    (1997) Mitochondrial toxins in models of neurodegenerative diseases. I: In vivo brain hydroxyl radical production during systemic MPTP treatment or following microdialysis infusion of methylpyridinium or azide ions. Brain Res 15:183–188.
    OpenUrl
  42. ↵
    1. Stout AK,
    2. Raphael HM,
    3. Kanterewicz BI,
    4. Klann E,
    5. Reynolds IJ
    (1998) Glutamate-induced neuron death requires mitochondrial calcium uptake. Nat Neurosci 1:366–73.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Tymianski M,
    2. Charlton MP,
    3. Carlen PL,
    4. Tator CH
    (1993) Source specificity of early calcium neurotoxicity in cultured embryonic spinal neurons. J Neurosci 13:2085–104.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Vannucci RC,
    2. Brucklacher RM,
    3. Vannucci SJ
    (1996) The effect of hyperglycemia on cerebral metabolism during hypoxia-ischemia in the immature rat. J Cereb Blood Flow Metab 16:1026–1033.
    OpenUrlCrossRefPubMed
  45. ↵
    1. Villalba M,
    2. Martinez-Serrano A,
    3. Gomez-Puertas P,
    4. Blanco P,
    5. Borner C,
    6. Villa A,
    7. Casado M,
    8. Gimenez C,
    9. Pereira R,
    10. Bogonez E
    (1994) The role of pyruvate in neuronal calcium homeostasis. Effects on intracellular calcium pools. J Biol Chem 269:2468–2476.
    OpenUrlAbstract/FREE Full Text
  46. ↵
    1. Wang GJ,
    2. Thayer SA
    (1996) Sequestration of glutamate-induced Ca2+ loads by mitochondria in cultured rat hippocampal neurons. J Neurophysiol 76:1611–1621.
    OpenUrlPubMed
  47. ↵
    1. Wieloch T
    (1985) Hypoglycemia-induced neuronal damage prevented by an N-methyl-d-aspartate antagonist. Science 230:681–683.
    OpenUrlAbstract/FREE Full Text
  48. ↵
    1. Zasslow MA,
    2. Pearl RG,
    3. Shuer LM,
    4. Steinberg GK,
    5. Lieberson RE,
    6. Larson CP
    (1989) Hyperglycemia decreases acute neuronal ischemic changes after middle cerebral artery occlusion in cats. Stroke 20:519–523.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Email
Print
View Full Page PDF
Citation Tools
Respond to this article
Neuroprotective Effect of High Glucose Against NMDA, Free Radical, and Oxygen–Glucose Deprivation through Enhanced Mitochondrial Potentials
So Y. Seo, Eun Y. Kim, Harriet Kim, Byoung J. Gwag
Journal of Neuroscience 15 October 1999, 19 (20) 8849-8855; DOI: 10.1523/JNEUROSCI.19-20-08849.1999
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords

Responses to this article

Respond to this article

Jump to comment:

No eLetters have been published for this article.