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Keywords:

  • cerebral ischaemia;
  • free radical;
  • hypothermia;
  • neuroprotection;
  • nitric oxide

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Effects of hypothermia on oxygen–glucose deprivation-induced neurodegeneration
  6. Mechanism of hypothermic neuroprotection
  7. Discussion
  8. References

Hypothermia has been demonstrated to be an effective neuroprotective strategy in a number of models of ischaemic and excitotoxic neurodegeneration in vitro and in vivo. Reduced glutamate release and free radical production have been postulated as potential mechanisms underlying this effect but no definitive mechanism has yet been reported. In the current study, we have used oxygen–glucose deprivation in organotypic hippocampal slice cultures as an in vitro model of cerebral ischaemia. When assessed by propidium iodide fluorescence, reducing the temperature during oxygen–glucose deprivation to 31–33°C was significantly neuroprotective but this effect was lost if the initiation of hypothermia was delayed until the post-insult recovery period. The neuroprotective effects of hypothermia were associated with a significant decrease in both nitric oxide production, as assessed by 3-amino-4-aminomethyl-2′,7′-difluorofluorescein fluorescence, and superoxide formation. Further, hypothermia significantly attenuated NMDA-induced nitric oxide formation in the absence of hypoxia/hypoglycaemia. We conclude that the neuroprotective effects of hypothermia are mediated through a reduction in nitric oxide and superoxide formation and that this effect is likely to be downstream of NMDA receptor activation.

Abbreviations used
DAF-FM

3-amino-4-aminomethyl-2′,7′-difluorofluorescein

DHEt

dihydroethidium

l-NAME

NG-nitro-l-arginine-methyl ester

NO

nitric oxide

NOS

nitric oxide synthase

OGD

oxygen–glucose deprivation

OHSC

organotypic hippocampal slice culture

PI

propidium iodide

SFM

serum-free medium

It has long been established that mild (36–34°C) to moderate (33–31°C) hypothermia has beneficial effects on outcome in a variety of brain injuries and the reduction of body temperature has been used for some time to prevent brain damage during cardiac surgery (Lougheed et al. 1955; Williams and Spencer 1958) and in patients with severe head injuries (Marion 2002). Recently there has been renewed interest in the possibility of using moderate reductions in brain temperature to prevent secondary damage following traumatic brain injury and stroke (Clifton et al. 1991; Colbourne et al. 1997; Corbett and Thornhill 2000). This renewed interest has arisen partly because of the impressive degree of neuroprotection observed in animal models of ischaemia when hypothermia is applied during the insults and partly because of the realization that long-term improvements can now also be achieved by prolonged post-ischaemic cooling in rat models of focal and global ischaemia (Colbourne and Corbett 1995; Yanamoto et al. 1996).

It is likely that the neuroprotective effects of hypothermia are mediated through a number of intra- and intercellular mechanisms including decreased rate of cellular ATP depletion (Erecinska et al. 2003), reduction in cytosolic free calcium (Kristian et al. 1992) and reduced free radical production (Kil et al. 1996). A reduction in the concentration of extracellular glutamate has also been reported in both stroke patients (Berger et al. 2002) and experimental ischaemia in rodents (Mitani and Kataoka 1991) although no change in glutamate was reported in a rodent model of traumatic brain injury (Palmer et al. 1993). However, the precise mechanisms underlying hypothermia-mediated protection are currently unclear. Most of the studies to date that have demonstrated protective effects of hypothermia have relied upon the use of animal models in which insults are performed at specific temperatures or where the brain is rapidly cooled after insults. While such animal studies are useful in devising optimal protective strategies based on hypothermia and determining long-term functional outcome, it is difficult to determine which cellular mechanisms might be involved in the neuroprotective effects using in vivo models. The dissociation of the effects of temperature on vascular and direct cellular actions is difficult to achieve in vivo. In vitro systems do have the advantage that direct cellular effects of hypothermia can be studied in the absence of any confounding vascular effects. So far only a limited number of studies have examined the effects of temperature on ischaemic neurodegeneration using in vitro preparations. Acute slices have been successfully used to study the functional recovery of synaptic transmission following oxygen and glucose deprivation at different temperatures (Tanimoto and Okada 1987; Taylor and Weber 1993; Greiner et al. 1998). Although it is possible to infer effects on cell survival from these experiments they cannot be used to reliably predict long-term neuroprotective effects because the survival times of most slice preparations are limited to a few hours. A number of studies have investigated the neuroprotective effects of hypothermia using cell culture models of excitotoxicity (Zeevalk and Nicklas 1993; Bruno et al. 1994; Berman and Murray 1996; Tymianski et al. 1998) and oxygen–glucose deprivation (OGD) (Bruno et al. 1994; Popovic et al. 2000). Although a reduction in neuronal loss was observed in all of these studies, there was no consensus on potential underlying mechanisms. One reason for this may be that the preparation of dissociated cell cultures disrupts neuronal connectivity and neuronal–glial intercellular communication therefore altering the way neurones respond to injury and hypothermia in culture. One in vitro method that overcomes many of these problems is the use of organotypic hippocampal slice cultures (OHSCs). A number of groups have used OHSCs to investigate the mechanisms underlying ischaemic injury (Strasser and Fischer 1995; Pringle et al. 1997, 2000; Zimmer et al. 2000; Cater et al. 2001; Gray et al. 2001) and it has been demonstrated that mild hypothermia reduces neuronal death in OHSC models of submersion ischaemia (Frantseva et al. 1999) or traumatic brain injury (Adamchik et al. 2000). However, neither of these studies attempted to investigate the mechanisms underlying the neuroprotective actions of ischaemia. In the current study, we have investigated the potential neuroprotective actions of hypothermia in an OGD model of cerebral ischaemia in OHSCs and the effects of hypothermia on free radical production. Our data demonstrate that mild hypothermia (31–33°C) during the period of OGD significantly reduces neuronal death and this is linked to a reduction in both superoxide and, in particular, nitric oxide (NO) production.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Effects of hypothermia on oxygen–glucose deprivation-induced neurodegeneration
  6. Mechanism of hypothermic neuroprotection
  7. Discussion
  8. References

Organotypic hippocampal slice cultures were prepared as described previously (Stoppini et al. 1991; Pringle et al. 1997). Neonatal Wistar rats (8–10 days old) were killed by decapitation and the hippocampus was rapidly dissected out. Transverse sections (400 µm) were cut on a McIlwain tissue chopper and placed into ice-cold Gey's balanced salt solution supplemented with 4.5 mg/mL glucose. Slices were plated onto sterile, semiporous tissue culture inserts (Millicell-CM; Millipore Corporation, Bedford, MA, USA; four slices per well) and maintained in culture at 37°C in a 5% CO2 atmosphere. The support medium consisted of 50% minimum essential medium, 25% Hank's balanced salt solution and 25% heat-inactivated horse serum supplemented with 4.5 mg/mL glucose and 1 mmol/L glutamine. The medium was changed every 3 days and cultures were used for experiments after 14 days in vitro.

All cultures were transferred into 1 mL of serum-free medium (SFM; 75% minimum essential medium, 25% Hank's balanced salt solution, 4.5 mg/mL glucose, 1 mmol/L glutamine) containing propidium iodide (PI, 5 µg/mL) 30 min prior to experimentation. In vitro ischaemia was modelled by combined OGD as described previously (Pringle et al. 1997, 2000) by placing cultures in serum- and glucose-free medium (100% minimum essential medium, 1 mmol/L glutamine) containing PI (5 µg/mL) for 60 min (previously saturated with 5% CO2 and 95% N2 by bubbling for 10 min and equilibrated to the required temperature). Culture plates were then placed in a modular incubator chamber that had previously been equilibrated to the appropriate temperature. The chamber was then flushed with 5% CO2/95% N2, sealed and placed in an incubator at the specified temperature. After 60 min, cultures were returned to normoxic SFM (+ 5 µg/mL PI), equilibrated to the required temperature and returned to an incubator for the 48-h post-insult recovery period.

Cell death was monitored by uptake of the fluorescent exclusion dye PI. This highly polar molecule is normally excluded from healthy cells with an intact plasma membrane but it gains access to the intracellular compartment of damaged cells and, upon binding to DNA, renders the nucleus highly fluorescent. The extent of PI fluorescence was determined as described previously (Morrison et al. 2002). Briefly, a transmission image was recorded prior to experimentation using an ORCA CCD camera (Hamamatsu Photonics UK Ltd, Welwyn Garden City, UK) on a DMIRBE inverted microscope (Leica, Milton Keynes, UK) and PI fluorescence images were recorded 24 and 48 h post-OGD using a standard rhodamine filter set (N2; Leica; excitation 515–560 nm, emission long pass 590 nm). The degree of neuronal damage was determined using Openlab 2.0 (Improvision, Warwick, UK). The percentage damage was determined by calculating the percentage of the area of the CA1 region in which PI fluorescence occurred above background, a measure which has been shown to correlate with the number of neurones which have died (Newell et al. 1995). After imaging at the 48 h time point, cultures were fixed in 4% paraformaldehyde and stained with thionin in order to histologically confirm damage.

The OGD-induced production of superoxide radicals was assessed by fluorescence imaging using dihydroethidium (DHEt) (Budd et al. 1997). Cultures were loaded by incubation in 500 µL SFM containing 15 µmol/L DHEt (Molecular Probes, Leiden, Holland). Fluorescence imaging was performed on an IMT-2 inverted microscope (Olympus, Southall, UK) equipped with a polychrome II argon lamp (TILL Photonics, Grafelfing, Germany) and data were collected using an Axon Imaging Workbench 2.1 data acquisition system (Axon Instruments, Egham UK) equipped with a Sensi-Cam CCD camera (PCO Imaging, Banbury, UK). The temperature of the stage was determined using a Peltier-controlled stage microincubator (Digitimer Ltd, Welwyn Garden, City, UK). Slice cultures were secured inside the recording apparatus and continually perfused with oxygenated (95% O2/5% CO2) PIPES buffer of the following basic composition (in mm): CaCl2, 5; KCl, 5; MgCl2, 2; NaCl, 120; PIPES, 20 and glucose, 20; pH 7.0 with NaOH. The OGD was initiated by switching the perfusate to deoxygenated glucose-free PIPES buffer (as above, with glucose substituted by sucrose, deoxygenated with 95% N2/5% CO2) In addition, humidified 95% N2/5% CO2 was passed across the cultures within the stage microincubator. Six individual pyramidal neurones within the CA1 were regarded as individual regions of interest by drawing around the neuronal cell bodies with the use of Axon Imaging Workbench software (Axon Instruments). Each experimental repeat (n = 1) consisted of the mean pixel intensity of six regions of interest captured at 5-min intervals. The mean percentage change in fluorescence above control was calculated using the formula [(F − Fo)/Fo] − [(Fc − Fco)/Fco]*100 where F is fluorescence intensity, Fo is mean fluorescence intensity prior to insult (images 1–5), Fc is mean fluorescence intensity of control and Fco is initial mean fluorescence intensity of control (images 1–5).

Nitric oxide production during OGD was assessed by fluorescence imaging using 3-amino-4-aminomethyl-2′,7′-difluorofluorescein (DAF-FM) as previously described (Morrison et al. 2002). Cultures were loaded by incubating for 2 h in 500 µL of SFM containing 20 µmol/L DAF-FM. Cultures were then placed in SFM without dye and returned to the incubator for 60 min before the DAF-FM fluorescence was recorded. Immediately following imaging, 60 min OGD at either 31 or 37°C was initiated as previously described. The DAF-FM fluorescence was then recorded immediately post-OGD using a monochrome video camera (COHU, San Diego, CA, USA) connected to an Apple Macintosh computer, saved and analysed offline using Scion Image (Scion Corp., Frederick, MD, USA).

The pixel intensity of the CA1 region was analysed by drawing regions of interest around this hippocampal substructure with Scion Image. The mean percentage change in fluorescence was calculated using the formula {[(F − Fo)/Fo]*100} where F is fluorescence intensity following OGD exposure and Fo is fluorescence intensity prior to insult. The average change in fluorescence in the CA1 region of each of the four cultures on a Millicell insert prior to the 60-min period of OGD was calculated (n = 1) and determined again post-OGD.

To investigate the effects of temperature on the temporal profile of NO production, we devised a system to continuously monitor the production of NO using confocal microscopy of cultures exposed to NMDA toxicity at 31 or 37°C. Cultures were loaded with DAF-FM as described above. PIPES buffer maintained at either 31 or 37°C was oxygenated (95% N2/5% CO2) for 20 min prior to imaging. Culture wells were taken out of the incubator, secured into a small Petri dish and 2 mL of oxygenated PIPES was added in a Peltier-controlled stage microincubator maintained at 31 or 37°C. The stage microincubator was mounted on an upright microscope (IMT-2; Olympus) fitted with a 60 × objective configured as a confocal laser-scanning microscope fitted with an argon/krypton laser (MRC1024ES; Biorad, Hemel Hempstead, UK). Images were taken at 10-min intervals prior and during exposure to 500 µmol/L NMDA. NMDA was added directly to the Petri dish in order to produce a final concentration of 500 µmol/L. Images were acquired with the use of a 60 × objective. These images were analysed with the use of Scion Image to calculate the mean pixel intensity within the whole field of view, this field of view was then regarded as the region of interest. The mean percentage change in fluorescence above control was calculated using the equation [(F − Fo)/Fo] − [(Fc − Fco)/Fco]*100 where F is fluorescence intensity, Fo is mean fluorescence intensity prior to insult (images 1 and 2), Fc is fluorescence intensity of control and Fco is initial fluorescence intensity of control (images 1 and 2).

Materials were obtained from the following suppliers: tissue culture plates from Nunc, Fisher Scientific (Loughborough, UK), all other tissue culture plastics from Bibby Sterilin (Stone, UK), culture media from Invitrogen (Paisley, UK), PI and DHEt from Molecular Probes, NG-nitro-l-arginine-methyl ester (l-NAME) from Tocris (Bristol, UK), DAF-FM and 2,2,6,6-tetramethylpiperidine-N-oxyl from Calbiochem (CNBiosciences, Nottingham, UK) and all other chemicals from Sigma Aldrich (Poole, UK).

Effects of hypothermia on oxygen–glucose deprivation-induced neurodegeneration

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Effects of hypothermia on oxygen–glucose deprivation-induced neurodegeneration
  6. Mechanism of hypothermic neuroprotection
  7. Discussion
  8. References

We compared the effects of hypothermia on OGD-mediated neuronal death when the temperature was reduced during the period of OGD or during the subsequent 48-h recovery period. Little or no PI fluorescence was observed in OHSCs maintained at 37°C for 48 h in the absence of OGD. In cultures exposed to 60 min OGD under normothermic conditions (37°C during both the OGD and recovery periods), significant PI fluorescence was observed at 24 h which became more intense 48 h after OGD. As reported previously (Newell et al. 1995; Strasser and Fischer 1995; Pringle et al. 1997), PI fluorescence was most prominent in the CA1 region with fluorescence also observed in the granule cell layer of the dentate gyrus and CA3/4 pyramidal neurones. For the hypothermia studies, data were normalized to the damage observed 48 h after 60 min OGD. We first investigated the effects of altering the temperature during the OGD insult. Reducing the temperature to 35°C had no effect on OGD-induced neurodegeneration either 24 or 48 h after the insult. However, when the temperature was reduced to 33 or 31°C, a statistically significant reduction in PI fluorescence was observed (Fig. 1a).

image

Figure 1. (a) Neuroprotective effects of hypothermia on damage in the CA1 region of hippocampal slice cultures. Cultures were exposed to a 1-h ischaemic insult by incubation in oxygen and glucose-free medium at different temperatures and then returned to normoxic medium containing glucose for 48 h. The degree of cell death was estimated by uptake of the fluorescent vital dye propidium iodide (PI) 24 (▪) and 48 h (bsl00023) after the insult and expressed as the area of CA1 in which PI fluorescence was detected. All data are normalized to the reference value, i.e. 48 h after 1 h of ischaemia at 37°C. Error bars represent the SEM. Post-hoc Student's t-test *p < 0.05, ***p < 0.001 preceded by a one-way analysis of variance (d.f. 61, f = 6.0, p = 0.00042). (b) PI fluorescence image of an organotypic hippocampal slice culture 48 h post-oxygen–glucose deprivation (OGD) at 27°C demonstrating strong fluorescence particularly in the CA1 region (scale bar, 1 mm). (c) In contrast, PI fluorescence is virtually absent 48 h after OGD performed at 31°C.

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When assessed 24 h after OGD, there was a reduction of 57% in PI fluorescence at 33°C and 94% at 31°C compared with OGD performed at 37°C and this was maintained 48 h post-OGD (73% protection, 33°C; 94% protection, 31°C). Examples of PI images are shown in Figs 1(b and c) demonstrating the reduction in PI fluorescence observed when OGD was performed at 31 compared with 37°C. In order to determine whether the reduction in PI fluorescence was due to a reduction in neuronal death rather than an effect of reduced temperature on the behaviour of PI itself, cultures were fixed after imaging and stained with thionin, a Nissl stain. In cultures exposed to OGD at 37°C, neurones in the CA1 region appeared as small, darkly stained pyknotic cells indicating neuronal death. In contrast, in those cultures exposed to OGD at 31°C neurones in the CA1 region had large lightly stained nuclei surrounded by more darkly staining cytoplasm, indicative of healthy neurones and confirming that intraOGD hypothermia was potently neuroprotective (data not shown).

A second set of experiments was performed to investigate the effects of delaying the onset of hypothermia until the post-insult recovery period. As 31°C was highly neuroprotective in the intrainsult paradigm, this temperature was used for the post-insult experiments. When assessed 48 h post-OGD, cultures in which the intraOGD temperature was 31°C and the recovery temperature was 37°C had significantly less (84%) PI fluorescence than those in which the OGD was performed at 37°C and the recovery period was at 37°C, replicating the results described above. However, no neuroprotection was observed when cultures were subjected to OGD at 37°C and then immediately placed into hypothermic (31°C) medium immediately after the end of the period of OGD for 6, 12, 24 or 48 h (Fig. 2).

image

Figure 2. Lack of effects of post-insult hypothermia on neuronal survival in cultures exposed to 1 h of oxygen–glucose deprivation (OGD) (bsl00023) compared with hypothermia during the insult (▪). The degree of cell death was estimated by uptake of the fluorescent vital dye propidium iodide (PI) 48 h after the insult. Hypothermic treatment at 31°C for 6, 12, 24 or 48 h after the insult was not neuroprotective. All data are normalized to the reference value 48 h after 1 h of ischaemia at 37°C (□); mean ± SEM, n = 8–12, ***p < 0.001 versus control.

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Mechanism of hypothermic neuroprotection

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Effects of hypothermia on oxygen–glucose deprivation-induced neurodegeneration
  6. Mechanism of hypothermic neuroprotection
  7. Discussion
  8. References

The production of free radicals has been strongly implicated in the pathogenesis of ischaemic neurodegeneration (Siesjo et al. 1999; Stewart and Heales 2003). We therefore decided to investigate the effects of hypothermia on free radical production induced by OGD in OHSCs.

Using DHEt imaging, we found that base levels of superoxide rose steadily over time which is thought to represent the normoxic production of superoxide as a by-product of respiration. Due to this phenomenon, experimental repeats were conducted to calculate the mean change in fluorescence intensity during normoxic perfusion at each temperature and this was then subtracted from experimental repeats in which OGD was induced. This resulted in a stable baseline of fluorescence prior to induction of OGD. Fluorescence began to increase almost immediately upon exposure to OGD and reached a plateau 25–30 min after onset of OGD. The DHEt fluorescence remained relatively constant until the cessation of OGD, after which there was a decrease back to baseline levels (Fig. 3). When assessed by two-way analysis of variance using time and temperature as variables, hypothermia had a highly significant effect on DHEt and therefore superoxide production.

image

Figure 3. Mean percentage change in dihydroethidium (DHEt) fluorescence following oxygen–glucose deprivation (OGD) at 37°C (•) and 31°C (▪) relative to non-OGD control slices. Hypothermia had a highly significant effect on DHEt fluorescence (p < 0.001 for both temperature and time). Data are expressed as the mean ± SEM; n = 9 (31°C) and n = 5 (37°C).

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Nitric oxide formation during the standard OGD protocol was assessed by using the fluorescent dye DAF-FM. We determined the level of DAF-FM produced in cultures exposed to OGD at either 37 or 31°C immediately after the termination of OGD. In control cultures maintained at 37°C for 60 min in the absence of OGD no significant change in DAF-FM fluorescence was observed compared with pre-experiment baseline controls (Fig. 4a). In cultures exposed to OGD at 37°C, DAF-FM fluorescence was significantly increased above baseline by 17.0 ± 3.6% (p < 0.001, n =43) (Fig. 4a). In contrast, when OGD was performed at 31°C, DAF-FM fluorescence was only 1.1 ± 1.9 above baseline, significantly below that observed at 37°C (p < 0.001, n = 46).

image

Figure 4. (a) Mean percentage change in DAF-FM fluorescence immediately after 60 min oxygen–glucose deprivation (OGD) relative to baseline levels. No significant change was observed in cultures maintained at 37°C for 60 min in the absence of OGD (control, n = 30) but a highly significant increase in DAF-FM fluorescence occurred when OGD was performed at 37°C (OGD 37°C, n = 43; ***p < 0.001 vs. control) but not when OGD was performed at 31°C (OGD 31°C, n = 46; †††p < 0.001 vs. OGD 37°C). (b) Mean percentage change in DAF-FM fluorescence in cultures exposed to 500 µm NMDA for 30 min. At 37°C (solid line) DAF-FM fluorescence became significantly raised above baseline after 30 min in NMDA (*p < 0.05 vs. baseline, n = 14) but this was attenuated when NMDA exposure was performed at 31°C (broken line) (**p < 0.01 vs. 37°C, n = 8).

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One possible mechanism through which hypothermia may produce neuroprotective effects is an alteration of the post-synaptic response to glutamate (Shima et al. 2003). In order to determine whether this was a factor in the significant attenuation in NO production observed following hypothermic OGD, we investigated the effects of hypothermia on NMDA-mediated excitotoxicity in the absence of the metabolic stress of OGD. We were able to perform the experiment in cultures exposed to the excitotoxin NMDA at either 31 or 37°C. Exposure to 500 µmol/L NMDA for 30 min at 37°C resulted in a 116 ± 5% increase in DAF-FM fluorescence (n = 14). No change in DAF-FM fluorescence was observed in cultures exposed to NMDA at 31°C (p < 0.01 vs. NMDA-treated cultures after 20 and 30 min), supporting the observations made in the OGD model that neuroprotective hypothermia is linked to a marked reduction in NO formation (Fig. 4b).

In order to test the hypothesis that suppression of NO production is linked to reduced OGD-mediated neurodegeneration, we assessed the effects of the NO synthase (NOS) inhibitor l-NAME in the OGD model; 24 h after OGD performed at 37°C, PI fluorescence was observed in 42.1 ± 3.3% of the CA1 pyramidal cell layer (n = 6). In contrast, in the presence of 10 mmol/L l-NAME, PI fluorescence was only observed in 2.5 ± 1.4% of CA1 (n = 6, p < 0.001 vs. OGD alone) (Fig. 5), a reduction of over 90%. Similarly, a free radical scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl (500 µmol/L) was also significantly neuroprotective against OGD, reducing PI fluorescence by 50% (Fig. 5).

image

Figure 5. Neuroprotective effects of NG-nitro-l-arginine-methyl ester (l-NAME) and 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) in cultures exposed to 60 min oxygen–glucose deprivation (OGD). l-NAME (10 mm) or TEMPO (500 µm) were present 20 min pre-OGD, during the OGD and throughout the 24-h recovery period prior to propidium iodide fluorescence imaging. Both compounds were significantly neuroprotective against OGD-induced neurodegeneration (***p < 0.001; *p < 0.05). n = 6 for l-NAME experiment and n = 4 for TEMPO experiment.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Effects of hypothermia on oxygen–glucose deprivation-induced neurodegeneration
  6. Mechanism of hypothermic neuroprotection
  7. Discussion
  8. References

The present study demonstrates clearly that reducing the intraOGD temperature to 31–33°C has a significant neuroprotective effect compared with cultures exposed to OGD at 37°C. Virtually no cell death was detected in the CA1 region of hippocampal slice cultures exposed to OGD for 1 h at 31°C compared with exposure at 37°C, as shown by reduced PI uptake and histological evidence of reduced cell death in thionin-stained sections. An intermediate level of protection was seen if the OGD was carried out at 33°C and no significant neuroprotection was seen with insults carried out at 35°C. Hypothermia-mediated neuroprotection was long lasting with a robust reduction in neurodegeneration observed up to 48 h post-OGD. From our data we also postulate that hypothermia-mediated neuroprotection is related to a reduction in both NO and superoxide as we observed that both DAF-FM and DHEt fluorescence was significantly attenuated by hypothermia and that pharmacological blockade of NOS and a free radical scavenger were strongly neuroprotective.

The degree of neuroprotection and temperature dependence that we observed are similar to results reported for the effects of intraischaemic hypothermia on the reduction of infarct volume and hippocampal pyramidal cell loss in animal models of focal and global ischaemia (Busto et al. 1987; Buchan and Pulsinelli 1990; Dietrich et al. 1990; Welsh et al. 1990; Green et al. 1992; Ridenour et al. 1992; Ren et al. 2004). The temperature dependence of the hypothermic effects that we observed was strikingly similar to those reported by Frantseva et al. (1999) who induced OGD in OHSCs using a submersion model. In that study, OGD performed at 34°C was not protective but virtually no damage was observed if the OGD was performed at 31°C. Similarly, the same group reported that reducing the temperature to 31°C produced a long-lasting, significant reduction in neuronal loss in a model of traumatic brain injury in OHSCs (Adamchik et al. 2000). The robust neuroprotective effect that we observed in the OHSC model implies that mild hypothermia during the ischaemic insult exerts direct actions on neuronal tissue to confer a neuroprotective effect independent of the presence of a functional vascular compartment suggesting that anti-inflammatory and antipyretic effects of hypothermia are not involved (Coimbra et al. 1996; Kim et al. 1996; Toyoda et al. 1996; Dietrich et al. 1999). This is supported by the fact that compounds acting on non-vascular elements are also neuroprotective in other studies using OHSCs (Strasser and Fischer 1995; Pringle et al. 1997, 2000; Zimmer et al. 2000; Cater et al. 2001; Gray et al. 2001).

Unlike many in vivo studies, we found that delaying the onset of the period of hypothermia until the post-OGD recovery period was clearly not neuroprotective, even when hypothermia (31°C) was maintained for 48 h. This finding contrasts with the numerous reports suggesting beneficial effects of post-ischaemic hypothermia in animal models of both global and focal transient ischaemia (Colbourne and Corbett 1995; Yanamoto et al. 1996; Colbourne et al. 1997; Corbett and Thornhill 2000). Recent studies have however highlighted the fact that the more prolonged the post-ischaemic hypothermia, the more beneficial this is to the long-term functional and histological outcome (Maier et al. 1998; Yanamoto et al. 1999; Huh et al. 2000). Shorter periods of post-ischaemic hypothermia (less than 3 h) are generally not chronically neuroprotective and delaying the onset of hypothermia significantly reduces the neuroprotective effect (Colbourne and Corbett 1994; Dietrich et al. 1996; Yanamoto et al. 1996). Similarly, while significant protection is achieved with post-ischaemic hypothermia, it does not reach the same degree of neuroprotection as seen with intraischaemic hypothermia (Yanamoto et al. 1999). Previously, we have shown that post-ischaemic neuroprotection can be seen with glutamate receptor antagonists (Pringle et al. 1997; Morrison et al. 2002), indicating that it is possible to rescue these cultures after an ischaemic insult. It seems likely therefore that the critical element must be the timing of the hypothermia in relation to neurodegenerative mechanisms in ischaemia. This conclusion is supported by the observations of the lack of effects of post-ischaemic hypothermia on recovery of synaptic function in acute slices following oxygen and glucose deprivation. Greiner et al. (1998) demonstrated that induction of hypothermia following the induction of an anoxic depolarization was unable to restore function in acute hippocampal slices but, if hypothermia was induced during the insult, anoxic depolarization was prevented and synaptic function could be maintained. It seems possible therefore that a major effect of post-ischaemic hypothermia in transient ischaemia in vivo could be to limit the degree of secondary cell death linked with anoxic depolarizations and spreading depression (Nedergaard 1996).

Although the mechanisms underlying the neuroprotective effects of hypothermia are not fully understood, one likely possibility is that there is a modulation of excitotoxic processes. In many (Busto et al. 1989; Mitani and Kataoka 1991; Berger et al. 2002) but not all (Palmer et al. 1993) cases, a significant decrease in the extracellular concentration of glutamate in hypothermic conditions has been observed compared with normothermic conditions. However, it is unlikely that this reduction in glutamate is directly related to hypothermia-induced neuroprotection because artificial elevation of the glutamate concentration during hypothermia does not result in neurotoxicity (Yamamoto et al. 1999). Further, hypothermia is significantly neuroprotective in a number of models of direct excitotoxicity in the absence of ischaemia/hypoxia (Bruno et al. 1994; Berman and Murray 1996; Suehiro et al. 1999) where toxicity is produced by direct activation of the post-synaptic receptor. Taken together, these results suggest that the neuroprotective effect of hypothermia is related to a direct action on vulnerable neurones. This is supported by our data demonstrating that NO production following exposure to NMDA is significantly attenuated by hypothermia. As NMDA toxicity is not dependent on the synthesis, release or uptake of an endogenous neurotransmitter, the implication of these experiments is that NO production occurs downstream of NMDA receptor activation and that hypothermia acts to suppress this.

Our observation that hypothermia-mediated neuroprotection has an absolute requirement for the reduction in temperature to be present during the period of OGD suggests that the mechanism involved occurs early during the biochemical cascade initiated by OGD. Ischaemic neurodegeneration is initiated by a rapid and sustained depletion in ATP leading to loss of ionic homeostasis, mitochondrial dysfunction, free radical formation and cell death (Greene and Greenamyre 1996; Siesjo et al. 1999). Mild hypothermia has been demonstrated to prevent both ATP depletion and accumulation of intracellular sodium and calcium ions in in vivo models of ischaemia (Amorim et al. 1999; Taylor et al. 1999). Alternatively, it has been suggested that hypothermia may be neuroprotective by enhancing recovery of protein synthesis during the post-ischaemic period (Widmann et al. 1993), preserving protein kinase activity (Hu et al. 1995) and reducing free radical formation (Kil et al. 1996; Maier et al. 2002). It is likely that changes in all of these elements are interlinked as preventing the loss of ATP synthesis will significantly attenuate any further disruption of neuronal function. We found that NO production was attenuated by hypothermia and that there was a reduction in superoxide formation, although not to the same degree during OGD, demonstrating that hypothermia affects individual cellular processes to different degrees, at least at 31°C.

We observed a significant effect of hypothermia on OGD-induced superoxide radical formation consistent with observations from in vivo experiments where both superoxide and hydroxyl radical formation is suppressed by hypothermia during and after an ischaemic episode (Kil et al. 1996; Zhao et al. 1996; Maier et al. 2002). However, unlike many in vivo models (Siesjo et al. 1999), we observed elevated superoxide formation only during the period of OGD with no post-OGD, ‘reperfusion’-type burst of radical formation. The reason for this discrepancy is unclear, although it is possible that there is an intracellular re-distribution of the ethidium ion during mitochondrial repolarization which leads to increased mitochondrial ethidium concentration and fluorescence quenching (Budd et al. 1997). The effect on superoxide radical formation that we observed was smaller than that for NO, probably because the most profound action of hypothermia on superoxide in vivo occurs in the penumbral zone (Maier et al. 2002) and there is no equivalent of this region in our OHSC model. In order to determine whether the decrease in superoxide was related to the neuroprotective actions of hypothermia we determined that the free radical scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl was neuroprotective against OGD at 37°C. This observation is in line with a previous report (Barth et al. 1996) which demonstrated that a spin trap compound was neuroprotective in OHSCs exposed to OGD. It is therefore likely that at least part of the mechanism through which hypothermia acts to reduce OGD-induced neuronal death is related to a reduction in the production of superoxide, a mechanism which has been previously postulated from work in in vivo models of cerebral ischaemia.

Reduced NO formation under hypothermic conditions may occur for a number of reasons, including down-regulation of NOS, altered NOS function, different properties of DAF-FM at low temperature or altered coupling of NOS to intracellular signalling pathways. The expression of both inducible-NOS and neuronal-NOS is reduced by hypothermia (Han et al. 2002; Karabiyikoglu et al. 2003) following experimental ischaemia in vivo. It is unlikely that this is relevant in our model as NO production was determined immediately after the cessation of OGD and significant up-regulation of NOS would not be expected to occur during the 60-min period of OGD. DAF-FM was used as a fluorescent indicator of NO production. Studies in invertebrate preparations (Schuppe et al. 2002) have demonstrated that DAF-FM acts as a sensitive marker of NO formation at 22–24°C, far below the minimum temperature used in our study, implying that the reduced DAF-FM fluorescence that we observed at 31 compared with 37°C was not due to altered performance of the DAF-FM in hypothermic conditions. The direct catalytic activity of both neuronal-NOS and inducible-NOS is temperature dependent and NOS activity is reduced by approximately 50% when the temperature is reduced to 31°C (Venturini et al. 1999); this may explain the significant difference that we observed in NO production following exposure to both OGD and NMDA at 31°C. Neurotoxicity mediated through NMDA activation, such as that occurring during ischaemia, is mediated in part through activation of NOS (Iadecola 1997). The NMDA receptor interacts with NOS through post-synaptic density protein-95 and disruption of this interaction is neuroprotective (Sattler et al. 1999). Although there is no direct evidence to suggest that hypothermia affects this interaction, it should be considered as a possible mechanism through which NMDA-mediated NO production can be attenuated during hypothermia. It is therefore likely that the reduction in NO formation that we observed under hypothermic conditions is due to physiological effects rather than altered properties of DAF-FM and that reducing NO production is neuroprotective. This is supported by studies demonstrating a significant reduction in glutamate receptor-induced NO production either through direct measurement of NO concentration (Takei et al. 2004) or through indirect determination of NO-derived nitrate and nitrite products (Shima et al. 2003). Taken together, these data strongly support the hypothesis that the efficacy of the coupling between NOS and glutamate receptors is significantly reduced under hypothermic conditions leading to an almost total attenuation of NO production.

Our data imply that reduced NO and superoxide production is directly linked to hypothermia-induced neuroprotection. In order to test this hypothesis we investigated the effects of artificially reducing NO and superoxide production during OGD performed at 37°C. The pharmacological inhibition of NOS with l-NAME resulted in a complete reduction of OGD-induced neurodegeneration, demonstrating that a selective reduction in NO production is sufficient to be neuroprotective even at 37°C. Similarly, the free radical scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl was also neuroprotective although the degree of protection was markedly less than observed with l-NAME, suggesting an important contribution of free radicals to the degenerative process. Many studies have demonstrated that the attenuation of either NO- or free radical-linked processes is strongly neuroprotective in both in vivo and in vitro models of ischaemia (Iadecola and Alexander 2001) and therefore the association between hypothermia, neuroprotection and attenuated NO and free radical production is not surprising. It has recently been suggested that OHSCs are resistant to NO-mediated toxicity (Keynes et al. 2004), implying that the reduction in NO that we observed may not be critical in hypothermia-induced neuroprotection. However, those experiments were apparently performed at 33°C, the temperature at which hypothermia becomes significantly neuroprotective, and it is therefore likely that the lack of NO-mediated toxicity observed by Keynes et al. (2004) is due in part to the experimental temperature.

In conclusion, we report that moderate hypothermia is neuroprotective in a model of OGD in OHSCs but only if the temperature is reduced during the period of OGD. Further, this effect is mediated through a reduction in NO and superoxide production.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Effects of hypothermia on oxygen–glucose deprivation-induced neurodegeneration
  6. Mechanism of hypothermic neuroprotection
  7. Discussion
  8. References
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