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Keywords:

  • apoptosis;
  • hypoglycaemia;
  • neurotoxicity;
  • recovery;
  • striatum

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

Glucose deprivation provides a reliable model to investigate cellular responses to metabolic dysfunction, and is reportedly associated with permanent cell death in many paradigms. Consistent with previous studies, primary cultures of rat striatal neurones exposed to 24-h hypoglycaemia showed dramatically decreased sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) metabolism (used as a marker of cell viability) and increased TUNEL staining, suggesting widespread DNA damage typical of apoptotic cell death. Remarkably, restoration of normal glucose levels initiated a sustained recovery in XTT staining, along with a concomitant decrease in TUNEL staining, even after 24 h of hypoglycaemia, suggesting recovery of damaged neurones and repair of nicked DNA. No alterations in the levels of four DNA repair proteins could be detected during hypoglycaemia or recovery. A reduction in intracellular calcium concentration was seen in recovered cells. These data suggest that striatal cells do not die after extended periods of glucose deprivation, but survive in a form of suspended animation, with sufficient energy to maintain membrane potential.

Abbreviations used
ACSF

artificial cerebral spinal fluid

BER

base excision repair pathway

DMEM

Dulbecco's modifed Eagle's medium

DMSO

dimethylsulfoxide

EAA

excitatory amino acid

ERCC3

excision-repair cross-complementing gene 3

MAP2

microtubule-associated protein-2

MSH2

human MutS homolog

NER

nucleotide excision repair pathway

PBS

phosphate-buffered saline

PMS

phenazine methosulfate

TCA

tricarboxlic acid cycle

VGCC

voltage-gated calcium channels

XRCC1

X-ray repair cross-complementing group 1

XTT

sodium(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbxanilide

The phenomenon of hypoglycaemia-induced energy failure is of clinical importance, as it not only accounts for the neurological outcome of insulin overdose in diabetic patients, but is likely to contribute profoundly to the brain damage associated with ischaemia and neurodegenerative diseases (Rothman and Olney 1986; Choi 1988).

It has been reported that the brain is uniquely dependent on the availability of glucose because it is among the most metabolically active tissue and in contrast to most other tissues essentially all of this metabolism is derived from plasma glucose rather than alternative substrates (Mobbs et al. 2001). Glucose is the main substrate for ATP in aerobic metabolism, hence when glucose is removed from tissue a decrease in ATP is observed (Kahlert and Reiser 2000). ATP levels in neurones decrease rapidly during hypoglycaemia in both in vitro and in vivo models (Mattson et al. 1993; McGowan et al. 1995; Santos et al. 1996; Madl and Royer 1999).

According to current concepts of the mechanisms involved in hypoglycaemic-induced cell death, the decrease in ATP levels results in an insufficient amount of energy for the function of the sodium-potassium ATPase dependent pump (Na+/K ± ATPase pump). A depletion in ATP therefore causes a collapse of ion gradients and as a consequence the cell membrane depolarises and the voltage-dependent ion channels open (Harris et al. 1984; Martin et al. 1994; Szatkowski and Attwell 1994). An opening of the voltage-gated calcium channels causes an influx of calcium which increases intracellular calcium levels that then can trigger a multitude of events, such as the release of excitatory neurotransmitters (Garthwaite and Garthwaite 1986), activation of calcium-dependent kinases (Favaron et al. 1990) and phospholipases (Farooqui and Horrocks 1991), the formation of free-radical compounds, and the activation of the mitochondrial permeability transition pore (Taylor et al. 1999). In addition, glutamate receptor stimulation can cause the activation of sodium channels, which induces the release of further glutamate via the reverse transport of glutamate by the glutamate transporter (Taylor et al. 1995; Strijbos et al. 1996). As a result, this initiates the triggering of the apoptotic process and ultimately nuclear DNA is cleaved. However, as apoptosis is an energy-dependent process, it might be anticipated that the rate of apoptosis might be retarded under hypoglycaemic conditions.

It has been demonstrated that the re-introduction of glucose into a previously energy-deficient system is less beneficial to the survival of neurones than would be expected (Siesjo 1992; Tasker et al. 1992). After a period of hypoglycaemia in both in vitro and in vivo models excitatory amino acid (EAA)-receptor activation and EAA release has been shown to occur when the glucose content is replaced (Linden et al. 1987; Tasker et al. 1992). It has also been suggested that a re-introduction of glucose initiates the opening the mitochondrial permeability transition pore (MTP) and triggers the formation of reactive oxygen species (ROS) (Kristian and Siesjo 1996). Many studies have reported the detection of apoptosis being a predominant feature in formally glucose deprived tissue after the replacement of glucose (Lawrence et al. 1996; Yakovlev et al. 1997; Ferrand-Drake et al. 1999).

In adult neurones, DNA damage can be corrected efficiently by DNA repair mechanisms. There are two established predominant pathways involved in repairing DNA damage (Coudore et al. 1997); the nucleotide excision repair pathway (NER) which handles rather infrequent lesions (Setlow and Carrier 1967) and the base excision repair pathway (BER) (Lindahl 1971) which is essential for recovery from spontaneous DNA damage (Seeberg et al. 1995). Whether these pathways are activated during hypoglycaemia or subsequent re-introduction of glucose is not clear.

The striatum is very vulnerable to energy failure, both in hypoglycaemia (Auer et al. 1984; Wieloch et al. 1985), and in transient reversible ischaemia (DeGirolami et al. 1984). It has been hypothesised that glutamatergic afferent fibres (from the cerebral cortex) and dopaminergic afferents (from the substantia nigra) contribute to this vulnerability. However, there is evidence that primary cultures of rat striatal neurones, where these extrinsic inputs are not present, are relatively insensitive to glutamatergic excitotoxicity (McDermott and Morris, unpublished observations). An in vitro model was therefore designed to investigate the direct effect of hypoglycaemia on cultured striatal neurones. In particular, we aimed to assess the consequences of re-introduction of glucose on the viability of striatal cells in hypoglycaemic cultures, and to test the hypothesis that striatal neurones possess an inherent capacity to recover from hypoglycaemic insult.

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

All products used in cell culture were obtained from Life Technologies (Invitrogen, Paisley, UK) except Euthatal (Rhône Mérieux Ltd, Harlow, UK). XTT and fura-2 were obtained from Molecular Probes, Eugene, OR, USA and the antibody to detect MAP2 from Sigma, Poole, UK. XRCC1 and DNA polymerase-β antibodies were obtained from Laboratory Vision Corporation, Fremont, CA, USA, Neomarkers, and ERCC3 and MSH2 antibodies were obtained from Autogen Bioclear, Calne, UK.

Primary dissociated cell cultures

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

Pregnant Wistar rats (200–300 g) gestation day 17 (E17) were killed with an overdose of sodium pentobarbitone (Euthetal), administered intraperitoneally. The embryonic dorsal striata were dissected out and prepared for culture according to Simpson et al. (1994). Cells were dissociated in 2.5% trypsin solution for 45 min at 37°C, triturated in Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX I™ (1000 mg/L) supplemented with 20% horse serum and 5 μg/mL penicillin-streptomycin, and seeded at a density of approximately (0.15 × 106 cells/cm), onto either 8-well multichamber glass slides or 6-well plates successively coated with poly d-lysine (4 μg/mL) and mouse laminin (6 μg/μL). The cultures were incubated at 37°C and 5% CO2/95% O2 in a humidified incubator.

After 72 h the medium was changed to Neurobasal medium (which contains 25 mm glucose) with penicillin-streptomycin (5 μg/mL) and B27 supplement added. Every 3 days thereafter the medium was changed. Cultures between 14 and 18 days old were used for experimentation.

Hypoglycaemic and recovery conditions

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

Cultures subjected to hypoglycaemia were washed twice with DMEM (without glucose and sodium pyruvate) and Neurobasal medium was replaced with the glucose/sodium pyruvate-free medium. Normoglycaemic (control) cultures were treated the same with the equivalent DMEM which contained glucose (5 mm) and sodium pyruvate (1 mm). The duration of hypoglycaemia-mediated toxicity was dependent on experimental procedure. The recovery phase was determined by returning the glucose content back into the cultures, using the medium for normoglycaemic cultures. Again, normoglycaemic cultures received equivalent procedures. DMEM contains the following amino acids: l-arginine, 0.4 mm; l-cystine, 0.2 mm; l-glutamine, 4.0 mm; glycine, 0.4 mm; l-histidine, 0.2 mm; l-isoleucine, 0.8 mm; l-leucine, 0.8 mm; l-lysine-HCl, 0.8 mm; l-methionine, 0.2 mm; l-phenylalanine, 0.4 mm; l-serine, 0.4 mm; l-threonine, 0.08 mm; l-tryptophan, 0.08 mm; l-tyrosine, 0.4 mm; l-valine, 0.8 mm.

Cell viability assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

Cell viability was assessed using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and phenazine methosulfate (PMS) according to standard procedures. Briefly, the XTT (1 mg/mL)/PMS (1.53 μL/mL) reagent was dissolved in PBS, added to the medium and incubated at 37°C and 5% CO2/95% O2 in a humidified incubator, for 3 hours. Subsequently, the medium was pipetted into a 96-well plate and each sample, in triplicate was read spectrophotometrically at a wavelength of 450 nm. Background signals, obtained from wells containing XTT/PMS in medium not exposed to cells were subtracted from the sample readings.

TUNEL staining

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

The APOPDETECT™ kit (The Quantum Biotechnologies Group, Q-Biogene, Harefield, UK), designed to label the free 3′-OH DNA termini in situ with digoxigenin labelled and unlabelled nucleotides, was used according to the manufacturer's protocol.

Immunocytochemistry

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

Cells were fixed using ice-cold 4% paraformaldehyde in PBS, for 30 min. After two washes with PBS, blocking serum (PBS with 15% normal goat serum) was added to the cells for 1 hour. The blocking serum was removed and the primary antibody solution (PBS with various specific antibodies (0.2–2 μg/mL), 3% normal goat serum and 0.005% Triton-X100) was added over night at 4°C in a humidified chamber. The antibodies used (and dilutions) were: microtubule-associated protein 2 (MAP2) (Sigma, 1 μg/mL); X-ray repair cross-complementing gene 1 (XRCC1) (Laboratory Vision Corp., 0.2 μg/mL), excision repair cross-complementing gene 3 (ERCC3) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA, 2 μg/mL), DNA mismatch repair protein MutS homologue 2 (MSH2) (Santa Cruz, 2 μg/mL), DNA polymerase-β (Laboratory Vision Corp., 1 μg/mL), bax (1 μg/mL), cytochrome c (1 μg/mL), caspase-3 (1 μg/mL), and caspase-9 (2 μg/mL) – all Santa Cruz Biotechologies. The cells were washed for 5 min with PBS, three times and then were incubated with the secondary antibody solution (PBS with specific species biotinylated secondary antibody (1 μg/mL) and 15% normal goat serum) for 60 min at room temperature (17–19°C). Following a further three 5-min washes with PBS, the cells were incubated with an ABC reagent (prepared using manufacturer's protocol) for 60 min at room temperature. The cells were washed again three times for 5-min per wash with PBS and then incubated with the VIP solution (prepared using manufacturer's protocol) for 10–15 min at room temperature, until staining was visible under the light microscope.

After the cells were rinsed twice, once in PBS for 5-min and once in distilled water for a further 5-min, they were dehydrated and then immersed in histoclear overnight. Cells were preserved by mounting the slides with coverslips using histomount and then left to dry.

Fura-2 calcium measurements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

Cells were grown on poly d-lysine and laminin-coated coverslips within 6-well plates, treated according to the experimental design and loaded with fura-2AM [1 μL/mL; dissolved as a 1-mm stock in dimethylsulfoxide (DMSO)] for 30 min at 37°C and 5% CO2/95% O2 in a humidified incubator. Small sections of coverslip were placed on a recording bath within a bath filled with ACSF (approximately 1 mL). Constant perfusion (1 mL per min) with ACSF occurred during the recording at room temperature. Glucose-free ACSF was used with hypoglycaemic-treated cells. Fluorescence measurements on single cells were made using a microfluorimeter consisting of an inverted fluorescence microscope (Nikon diaphot) and a photomultiplier tube with a bialkali photocathode (McCarron and Muir 1999). The excitation wavelengths (340 and 380 nm, 7 nm bandpass) were provided by a PTI Deltascan (Photon Technology International Inc, East Sheen, London, UK) and the cell was illuminated every 10 ms for 8.5 ms with each wavelength. [Ca2+]c measurements were made therefore at a frequency of 50 Hz. The emitted light from fura-2 (510 nm) was directed onto the photomultiplier which was operating in photon counting mode. Rmin and Rmax were also determined from in vitro calibrations and decreased by 15% to adjust for cell viscosity (Poenie 1990). Baseline recordings were allowed to run for approximately 1 min before measuring the ratios. The following equation was used to calculate the concentration of intracellular calcium (Grynkiewicz et al. 1985):

  • image

Where R is the experimental ratio obtained, Rmin the ratio obtained from a solution of fura-2 lacking calcium, Rmax (8.0) the ratio obtained from a solution of fura-2 containing saturating Ca2+, sf2 the emission detected from 380 nm excitation from the calcium-free solution, sb2 the emission detected from 380 nm excitation of the Ca2+-containing solution and Kd (224 nm), is the dissociation constant of fura-2 for Ca2+.

Image analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

For each experimental culture 30 immunoreactive cells were measured from randomly chosen fields of view (five cells per field of view). The relative optical density values of staining over the cytoplasmic area (apoptotic mediators) or the nuclear area (DNA repair proteins) was measured, and the background staining (measured over adjacent unstained cells – generally a small percentage of the total number of cells, readily discriminated from labelled cells for all these antibodies) subtracted to obtain a mean value for specific immunoreactive labelling. All measurements were made by using Image NIH version 1.52 software (W. Rasband, NIH).

Statistics

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

Data were converted to percentages of control to parallel vehicle-treated cells (except TUNEL staining data). Statistical significance was assessed using two-way analysis of variance (anova) with post hoc Tukey's test for comparison between groups where two factors were involved. One-way anova with post hoc Fisher's test was used for single factor analysis. The non-parametric Mann–Whitney test was used for comparison within groups. The 95% confidence intervals were used for comparison between treatment groups and vehicle treatment (100%).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

There was a significant decrease in cell viability, as assessed by XTT staining, in cells treated with hypoglycaemia for 24, 48, 72 and 96 h relative to cells retained under normoglycaemic conditions. There was no significant effect of the duration of hypoglycaemia on the level of XTT staining (Fig. 1). However, when a shorter time point (4 h) was monitored, a smaller decrease in XTT staining was observed: staining as a percentage of normoglycaemic cultures: 4 h − 56 ± 14%; 24 h − 25 ± 10%, n = 5.

image

Figure 1. The effect of glucose removal for 24, 48, 72 and 96 h on cell viability as assessed by XTT staining. compared to normoglycaemic cultures. Data are presented as a percentage of XTT staining in normoglycaemic cultures incubated for 24 h after the onset of treatment (100%) and error bars represent SEM (n = 3). All durations of hypoglycaemia studied produce a significant decrease in XTT staining (two-way anova, F[1,16] = 105.1 significant effect of hypoglycaemia, p < 0.001, effect of time F[3,16] = 0.7, p = 0.6, non-significant; *p < 0.05 vs. 100%, 95% confidence intervals of the mean).

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Neuronal cultures exposed to 24-h hypoglycaemia invariably showed a significant decrease in XTT staining compared with normoglycaemic cultures. Some variation between experiments was noted in terms of the magnitude of the effect (Figs 1 and 2), but in every case a highly significant fall in XTT staining in was observed. However, when the glucose was replaced for 24 h, XTT staining dramatically increased to levels equivalent to those in normoglycaemic cultures (Fig. 2). Glucose replacement for a further 24 and 48 h sustained the increased levels of cell viability (Fig. 2). The XTT staining in normoglycaemic cultures decreased slightly 24 and 48 h after the onset of treatment, but not after 72 h.

image

Figure 2. The effect of glucose replacement in cultures exposed to 24 h of hypoglycaemia. In hypoglycaemic cultures the glucose was removed for 24 h and then replaced for either 24, 48 or 72 h. Cell viability was measured by XTT staining. Data are presented as a percentage of XTT staining in normoglycaemic cultures incubated for 24 h (100%) and error bars represent SEM (n = 5). Both glucose replacement and duration of glucose replacement produce significant effects (two-way anova, effect of glucose replacement, F[1,16] = 7.1, p < 0.05; effect of recovery time, F[3,16] = 6.2, p < 0.01). The XTT staining of hypoglycaemic cultures after 24 h (0 recovery period) is significantly decreased compared with normoglycaemic cultures (*p < 0.05 vs. 100%, 95% confidence intervals of the mean). After glucose replacement for 24, 48 and 72 h, cell viability significantly increases (one-way anova, F[3,8] = 8.2, p < 0.01). A slight but significant decrease in XTT staining is observed in normoglycaemic cultures 24 and 48 h after the medium change.

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The morphology of cultured striatal neurones exposed to normoglycaemia, hypoglycaemia, or hypoglycaemia followed by recovery, was assessed by immunocytochemical visualisation of the cytoskeletal protein MAP2. Neurones in normoglycaemic cultures were evenly spread with long dendrites making contact with each other (Fig. 3a). In contrast, neurones in hypoglycaemic cultures showed shortened, fragmented dendrites compared with normoglycaemic cultures (Fig. 3b). Clumps of cells were observed in hypoglycaemic cultures, suggesting that neurones had migrated towards each other. Fewer stained neurones were detected by immunocytochemistry in hypoglycaemic cultures, and it is assumed that profoundly sick neurones had become unstuck from the adhesive coating lining the culture chambers and hence were lost during processing. Neurones subjected to hypoglycaemia followed by re-introduction of glucose showed a morphology similar to normoglycaemic cells: extended dendrites were visible, and neurones were not clumped together (Fig. 3c).

image

Figure 3. Morphology of striatal neurones after 24 h of normoglycaemia (a, d); 24 h of hypoglycaemia (b, e); or 24 h of hypoglycaemia followed by 24 h of recovery (c, f). The dendrites in hypoglycaemic cultures are retracted or fragmented. In recovered cultures the dendrites have spread out connecting the remaining cells. Scale bar represents 15 μm (a–c) or 60 μm (d–f).

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As hypoglycaemic neurotoxicity reportedly involves apoptotic mechanisms, we sought evidence of apoptosis in striatal cultures exposed to hypoglycaemia. A significant increase in TUNEL staining was observed in cultures exposed to hypoglycaemia for 24 h when compared with normoglycaemic cultures (Fig. 4). Remarkably, when glucose was restored to the culture medium, TUNEL staining significantly decreased to normoglycaemic levels. After 48 and 72 h of recovery, TUNEL staining was still reduced in comparison with staining in hypoglycaemic cultures with no recovery (t = 0). A slight increase in TUNEL staining was detected in normoglycaemic cultures after 72 h in the presence of glucose compared with normoglycaemic cultures at earlier time points (Fig. 4).

image

Figure 4. The percentage of cells showing TUNEL staining in cultures exposed to 24 h of normoglycaemia or hypoglycaemia and subsequently either 24, 48 or 72 h of glucose replacement. Both treatment and time produce a significant effect on TUNEL staining (two-way anova, effect of glucose replacement, F[1,16] = 10.28, p < 0.01; effect of recovery time, F[3,16] = 10.81, p < 0.001). Cultures exposed to 24 h of hypoglycaemia show a significant increase in TUNEL staining compared with normoglycaemic cultures (*p < 0.001, Tukey pair-wise comparison). This staining is significantly reduced when glucose is re-introduced for 24 h (p < 0.001, Tukey pair-wise comparison), 48 h (p < 0.001, Tukey pair-wise comparison) and 72 h (p < 0.01, Tukey pair-wise comparison). Data are presented as a percentage of the total number of cells in culture, and error bars represent SEM (n = 4).

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Resting intracellular calcium levels in the cell soma were measured using fura-2. While resting intracellular calcium levels did not change during hypoglycaemia relative to normoglycaemia, there was a significant decrease in the levels of intracellular calcium in cells which had been hypoglycaemic for 24 h and then recovered for 24 h, compared with levels of normoglycaemic and hypoglycaemic cells (measured by the change in the fura-2 signal) (Fig. 5). Levels of intracellular calcium in cells that had been recovered for 48 h were not significantly different from cells that had been recovered for 24 h (Fig. 5).

image

Figure 5. Intracellular calcium levels in normoglycaemic cells, hypoglycaemic cells and recovered cells. In recovered cells, the glucose was removed for 24 h then replaced for either 24 or 48 h. There is no significant change in the levels of intracellular calcium in hypoglycaemic cells relative to levels measured in normoglycaemic cells after either 24 or 48 h (p = 0.3, Mann–Whitney test). However, in cells recovered in glucose for 24 h after 24 h hypoglycaemia, the intracellular calcium level is significantly decreased compared with normoglycaemic cells incubated for 48 h (*p < 0.05, Mann–Whitney test) and hypoglycaemic cells incubated for 48 h (*p < 0.05, Mann–Whitney test). There is no significant difference in intracellular calcium levels between cells that had been recovered for 24 or 48 h (p = 0.6, Mann–Whitney test). Data are represented as a percentage of cultures in normoglycaemic conditions for 24 h (100%), and error bars represent SEM (n = 10).

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To test the hypothesis that the decreased TUNEL staining, during recovery from hypoglycaemia, might reflect increased activity of DNA repair processes, we monitored the levels of four DNA repair proteins. For two of the four DNA repair proteins studied, an increase in their expression has been shown during periods of genomic restoration (Hermon et al. 1998; Belloni et al. 1999).

There was no change in the expression of the immunoreactivity for any of the DNA repair proteins studied. XRCC1, ERCC3, MSH2 and DNA polymerase-β immunoreactivity levels in hypoglycaemic cells, or cells which had been recovered from hypoglycaemia, did not differ from normoglycaemic levels (Table 1).

Table 1.  The expression of XRCC1, ERCC3, MSH2, DNA polymerase-β DNA repair protein in normoglycaemic, hypoglycaemic and recovered cells
Duration of recovery (h)XRCC1ERCC3MHS2DNA polymerase-β
NormoHypoNormoHypoNormoHypoNormoHypo
  1. In hypoglycaemic cultures the glucose was removed for 24 h and in recovered cells the glucose was removed for 24 h then replaced for either 0.5, 1, 4, 24, 48 or 72 h. There is no significant change in the expression of XRCC1, ERCC3, MSH2, DNA polymerase-βimmunoreactivity in recovered cells relative to hypoglycaemic cells (one-way anova, p = 1.0; one-way anova, p = 0.8; one-way anova, p = 0.8; one-way anova, p = 1.0, respectively). Data are presented as a percentage of expression in normoglycaemic cultures incubated for 24 h (100%).

0100108.3 ± 10.2100103.7 ± 3.1100100.9 ± 2.0100112.7 ± 5.4
0.5103.4 ± 3.097.6 ± 3.4104.4 ± 3.5104.0 ± 4.393.3 ± 15.5104.5 ± 5.5114.1 ± 14.4113.3 ± 10.6
1103.7 ± 3.199.5 ± 4.9105.5 ± 3.0102.1 ± 4.2102.1 ± 6.2103.5 ± 9.0111.4 ± 5.9110.6 ± 3.5
4100.2 ± 8.695.9 ± 1.695.7 ± 14.295.6 ± 6.2101.2 ± 1.9104.4 ± 5.2109.4 ± 5.0106.2 ± 8.7
24116.2 ± 25.5121.4 ± 27.7100.3 ± 3.0101.3 ± 9.3102.0 ± 3.3100.3 ± 1.4122.8 ± 31.9115.4 ± 37.9
48127.2 ± 42.2119.6 ± 29.4101.6 ± 6.8103.4 ± 7.2103.3 ± 1.2102. ± 1.1115.8 ± 24.7121.1 ± 50.5
72123.0 ± 32.1119.2 ± 30.0103.2 ± 6.9104.9 ± 10.4102.8 ± 1.599.2 ± 3.3114.2 ± 25.3113.5 ± 27.7

The level of expression of various proteins related to apoptosis was monitored in hypoglycaemic cultures. However, no significant changes in expression of caspase-3, caspase-9, cytochrome c or bax could be detected at any time point (Fig. 6).

image

Figure 6. The levels of immunoreactivity (-ir) for bax, cytochrome c, caspase-9 and caspase-3 in cultures exposed to 24 h of normoglycaemia or hypoglycaemia and subsequently either 24, 48 or 72 h of glucose replacement. Neither treatment nor time produced a significant effect on the levels of staining for any of the proteins (two-way anova). Data are presented as a percentage of the levels of staining in control cultures (normoglycaemic with recovery period = 0 h), and error bars represent SEM (n = 3).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References

The toxic effects of hypoglycaemia on neurones in brain areas such as the striatum are well known. We observed that hypoglycaemia caused a significant reduction in cell viability, as assessed by mitochondrial reduction of XTT, in striatal cultures after 24 h (Fig. 1). This is consistent with previous studies (Nakao et al. 1995; Williams et al. 1995; Calabresi et al. 1997) and suggests that an alteration in mitochondrial functioning has occurred by 24 h. It is interesting that cell viability does not reach zero in our experiments. Hence, there could be a specific type of neurone or neurones within the culture which are relatively resistant to hypoglycaemic cell damage, or else all cells are partially but not completely compromised. This can be observed in Fig. 3. For example, it is known that cholinergic interneurones are much less vulnerable to hypoglycaemia compared with medium spiny neurones (Calabresi et al. 1997). However, striatal cultures represent a relatively homogenous cell population (Simpson et al. 1994); 95% of the cell population within the cultures are the same cell type (medium spiny neurones).

We have not formally measured ATP levels in this study. However, it is known that ATP levels decrease rapidly when glucose is not present (Cheng et al. 1993; Mattson et al. 1993; McGowan et al. 1995; Santos et al. 1996; Geng et al. 1997; Madl and Royer 1999; Zeevalk and Nicklas 2000). However, it is also clear that ATP levels are not totally depleted even after extended time periods (Mattson et al. 1993; Geng et al. 1997; Zeevalk and Nicklas 2000). Hence, the residual mitochondrial reduction of XTT probably reflects some enduring mitochondrial function, and possibly ATP production, possibly due to TCA cycle alterations utilising alternative substrates such as glutamate.

After 24 and 48 h of hypoglycaemia no change in [Ca2+]i was seen in cells when compared with normoglycaemic cells (Fig. 5). While this is contrary to most other studies (Mattson et al. 1993; Harris et al. 1984; Knopfel et al. 1990; Cheng and Mattson 1991; Tekkok et al. 1999), the lack of change in [Ca2+]i during hypoglycaemia is consistent with other reports (Cobbold and Bourne 1984; Haworth et al. 1987; Williams et al. 1995), and suggests that intracellular calcium homeostatic mechanisms remain functional during energy deprivation in striatal neurones.

The experiments in this study suggest that hypoglycaemia decreases mitochondrial functioning (XTT staining). In addition, some signs of apoptosis are present (TUNEL staining) whereas other are not – no increases in immunoreactivity for Bax, cytochrome c, caspase-9 or caspase-3 were detected, suggesting that hypoglycaemia produces a neurotoxic effect with some characteristics of apoptosis. However, this neurotoxic effect is not produced by calcium. Data from Fig. 4 shows that TUNEL staining was markedly increased in cells subjected to 24 h of hypoglycaemia (Fig. 4) suggesting that glucose deprivation produces DNA fragmentation, a distinct marker of apoptosis. This has been repeatedly reported in many models, including in vitro and in vivo models of hypoglycaemia (Lawrence et al. 1996; Ferrand-Drake et al. 1999). The presence of TUNEL staining implies that the DNA has been nicked by specific endonucleases. It has been reported that calcium-independent endonucleases exist, for example, Dnase II (Barry and Eastman 1993), therefore the activity of DNA nicking enzymes in this model is consistent with the lack of change in intracellular calcium levels (Fig. 5). One would assume DNA nicking is a result of caspase activation. However, we have observed that caspase-3 inhibitor, Z-DEVD-FMK, produced no neuroprotective effect in hypoglycaemic cultures (C. J. McDermott and B. J. Morris, unpublished), and we detected no increase in the levels of caspase-3 after hypoglycaemia. This suggests that caspase-3 is not involved in the apoptotic pathway initiated by the removal of glucose. It is possible that DNA fragmentation under conditions of energy deprivation proceeds via a caspase-independent mechanism as it has been reported that ATP is required for caspase activation but not for DNA fragmentation (Leist et al. 1997).

Data from this study indicate a different type of cell dysfunction occurring in striatal cultures exposed to hypoglycaemia compared with the generally accepted hypothesis (Siesjo 1992). Most studies report irreversible cell death from an excessive concentration of intracellular calcium. However, this study shows that cells can recover from such an insult shown by both XTT staining and TUNEL staining.

Replacing the glucose in the culture medium after 24 h of hypoglycaemia, markedly increases the XTT staining of the cells (Fig. 2), compared with hypoglycaemic cultures without glucose replacement (Fig. 1). This strongly suggests an increase in cell survival that is prolonged and not a transient effect, as after 72 h of recovery the cell viability of these cultures is still at normoglycaemic levels (Fig. 2). It is also possible that the dramatic increase in hypoglycaemic cultures when glucose is replaced is partly associated with the removal of toxic elements, such as ROS, as well as the re-introduction of glucose. Figure 3(c), provides a morphological picture of recovery in surviving cells which have regained long processes and are more spread out compared with cells exposed to hypoglycaemia. This finding is in contrast to many studies which have reported biochemical, morphological and genetic alterations to cells upon the re-introduction of energy that are detrimental to cell survival (Goldberg and Choi 1993; Taylor et al. 1999). For example, during the recovery period following insulin-induced hypoglycaemia, membranes of hippocampal cells ruptured, and mitochondrial densities and condensation of the nucleus was observed (Auer et al. 1985). It is also well documented that during the recovery phase there is an increase in mitochondrial production of ROS, as a consequence of injured mitochondria (White and Reynolds 1996), and also a release of cytochome c causing DNA fragmentation (Fujimura et al. 1999).

Remarkably, TUNEL staining was reduced concomitantly with glucose replacement, suggesting that as energy sources are restored in the culture, this repairs the DNA fragmentation via energy-dependent repairing mechanisms (Fig. 4). A decrease in TUNEL staining was observed up to 72 h of recovery. This is in conflict with an in vivo study (Ferrand-Drake et al. 1999), where TUNEL staining induced by severe insulin-hypoglycaemia in hippocampal neurones was observed after 24 h and was markedly enhanced after 48 h of recovery. The differences could reflect either the complexity of the in vivo system, or distinct mechanisms operating in hippocampal as compared with striatal neurones.

For the fragmented DNA to be repaired following glucose re-introduction, it is likely that DNA fragmentation is incomplete. Glucose deprivation may have triggered Dnase proteins, such as endonucleases, to cleave the DNA but under low-energy conditions these proteins function less efficiently. This incomplete fragmentation may have signalled, upon recovery, the activation of DNA repair proteins. However, the levels of expression of the four different DNA repair proteins were not changed during 72 h of recovery (Table 1). Our previous work has shown that the amount of an antigenic peptide, immobilised onto membranes, is proportional to the immunochemical staining intensity obtained following visualisation with biotinylated secondary antibody, ABC complex and VIP peroxidase substrate kit, as used here. Our experience, as that of many other groups, is that, under appropriately controlled conditions, semi-quantitative immunocytochemistry is a reliable method to detect altered protein expression. Hence, we believe that the levels of these proteins are not affected by hypoglycaemia, although it is possible that other repair proteins are activated. Studies investigating the roles of DNA repair proteins, using different models of brain damage, have provided an ambiguous database of results. In some instances, the expression of particular DNA repair proteins have decreased following a metabolic insult (Liu et al. 1996; Fujimura et al. 1999), whereas in other systems expression has increased (Hermon et al. 1998; Belloni et al. 1999). A decrease in expression could signal that the cells are permanently damaged and will subsequently die. However, it remains to be investigated whether DNA repair protein induction is a part of a protective response, or is a sign of suffering cells. In addition, glucose deprivation is likely to affect nucleotide synthesis directly via the pentose phosphate pathway. The apparent DNA damage we detect by TUNEL staining during hypoglycaemia, and the apparent DNA repair after return to normoglycaemic conditions could conceivably relate to this action. Unexpectedly, intracellular calcium levels measured by fura-2 were significantly reduced following 24 h of recovery compared with normoglycaemic levels. Previous studies have reported a dramatic increase in [Ca2+]i levels after 24 h of glucose replacement (Taylor et al. 1999). In some studies, a decrease is observed but for only a few hours before the levels rise above normoglycaemic levels (Mattson et al. 1993; Tymianski et al. 1993), however, in this study even after 48 h of recovery, [Ca2+]i levels still had not exceeded normoglycaemic concentrations (Fig. 5). From this result, it is logical to suggest that the plasma membrane is intact and the cell is functioning efficiently after 24 h of hypoglycaemia. Is a reduction in [Ca2+]i during recovery a protective mechanism? It is possible that re-introduction of glucose, initiates a fast sequestration of any excess [Ca2+]i by endoplasmic reticulum (ER) and mitochondria as well as an increase in the activity in calcium-binding proteins?

It also is possible that the cell up-regulates its buffering systems during the initial stages of hypoglycaemia before the energy supply has fallen to a particular threshold. For example, the cell may detect, via some glucose-responsive receptor (Ashford et al. 1990), that the concentration of glucose has decreased, and signal an up-regulation of calcium-binding proteins such as calbindin-D28K. In this system there is no calcium influx, during hypoglycaemia (Fig. 7) so the calcium-binding proteins have nothing to buffer and with ATP levels potentially declining, an inhibition of protein synthesis activity occurs. Subsequently, during the recovery period, perhaps as ATP levels increases, protein synthesis is initiated and the up-regulation of calcium buffering systems causes an unregulated extensive sequestration, or extrusion of calcium out of the cell and the [Ca2+]i is dramatically reduced.

image

Figure 7. A hypothesis of potential neuroprotective mechanisms during glucose deprivation. Both EAA receptors (EAA-R) and voltage-gated calcium channels (VGCC) are closed. With decreased energy supply, alternative substrates are utilised, such as glutamate which enters the tricarboxlic acid cycle (TCA) and provides sufficient amount of energy to maintain resting potential of the plasma membrane. Many components of the apoptotic response are prevented due to the decreased energy supply. However, some apoptotic factors are released, such as cytochome c, and DNA fragmentation occurs due to the activation of DNA endonucleases by stimuli, such as reactive oxygen species or calpains.

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Overall, these results suggest that a decrease in XTT staining and an increase in TUNEL staining does not signify death as affected cells can recover function and DNA integrity when glucose is replaced. The mechanisms of DNA repair remain to be elucidated, this may involve different DNA repair proteins or completely unidentified processes which regenerate DNA structure in cells not destined to die.

It is clear that a new hypothesis is required that is consistent with the data obtained in this study. It was previously reported that during hypoglycaemia the mitochondria are not irreversibly damaged. This suggests that mitochondria shut down temporarily whilst there is a reduction in glucose, perhaps to preserve energy levels, or perhaps there is insufficient amount of energy for the mitochondria to function effectively, therefore respiration becomes suspended. This may render the cell not technically dead but electrically quiescent. Suspended animation could resemble a hypothermic state. It is well established that hypothermia protects against delayed neuronal death following ischaemia (Busto et al. 1989). It is thought that the mechanism of this protective effect is associated with a slowing of the cells' metabolism by inhibiting oxygen and glucose consumption in the brain (Bering 1974) and by producing a ‘membrane-stabilising’ effect, perhaps reducing the Na+/K+ leakage. This could explain the increase in XTT staining following re-introduction of glucose.

The new hypothesis is as follows: when the extracellular source of glucose is removed, the activity of the TCA cycle decreases and ATP levels fall. The membrane remains at resting potential, VGCC and EAA receptors are closed and therefore synaptic transmission is suppressed. The ATP source required to maintain resting potential is provided by existing concentration of ATP left after glucose removal and alternative substrates such as glutamate (Fig. 7). The cell falls into a ‘suspended animation’ state where energy demand is reduced but with sufficient amount of energy to keep the membrane polarised but not to initiate active processes, namely apoptosis. However, this disruption of internal energy homeostasis triggers ATP-independent mechanisms resulting in the activation of endonucleases and fragmentation of DNA. During early hypoglycaemia, when energy levels are still relatively high, genes are up-regulated in the event of further cellular damage, such as calcium-buffering proteins and possibly DNA repair systems. However, following transcription of these genes the cell's energy begins to fall below a specific threshold and consequently these new proteins are not utilised until energy levels rise. On the re-introduction of glucose, ATP levels rise and cytosolic calcium is buffered via the previously up-regulated calcium buffering genes, metabolism within the mitochondria begins to function normally, all respiratory processes normalise, DNA repair begins, and hence, normal cellular functioning ensues.

The revised hypothesis could be termed a neuroprotective mechanism employed in striatal neurones against energy deprivation. It is reasonable to suggest that neurones, because of their post-mitotic nature require complex protective mechanisms, and the ability to enter a state of a suspended animation is possibly one of them.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Primary dissociated cell cultures
  6. Hypoglycaemic and recovery conditions
  7. Cell viability assay
  8. TUNEL staining
  9. Immunocytochemistry
  10. Fura-2 calcium measurements
  11. Image analysis
  12. Statistics
  13. Results
  14. Discussion
  15. Acknowledgements
  16. References
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