Phosphorylation of Extracellular Signal-Regulated Kinase after Transient Cerebral Ischemia in Hyperglycemic Rats

Neurobiology of Disease 8, 127–135 (2001) doi:10.1006/nbdi.2000.0363, available online at http://www.idealibrary.com on

Phosphorylation of Extracellular Signal-Regulated Kinase after Transient Cerebral Ischemia in Hyperglycemic Rats Ping-An Li, Qing Ping He, Ouyang Yi-Bing, Bing Ren Hu, and Bo K. Siesjo¨ Center for the Study of Neurological Disease, The Queen’s Medical Center, Honolulu, Hawaii Received June 20, 2000; revised September 22, 2000; accepted October 20, 2000

The present study was undertaken to investigate whether extracellular signal-regulated kinase (ERK) was involved in mediating hyperglycemia-exaggerated cerebral ischemic damage. Phosphorylation of ERK 1/2 was studied by immunocytochemistry and by Western blot analyses. Rats were subjected to 15 min of forebrain ischemia, followed by 0.5, 1, and 3 h of reperfusion under normoglycemic and hyperglycemic conditions. The results showed that in normoglycemic animals, moderate phosphorylation of ERK 1/2 was transiently induced after 0.5 h of recovery in cingulate cortex and in dentate gyrus, returning to control values thereafter. In hyperglycemic animals, phosphorylation of ERK 1/2 was markedly increased in the cingulate cortex and dentate gyrus after 0.5 h of recovery, the increases being sustained for at least 3 h after reperfusion. Hyperglycemia also induced phosphorylation of ERK 1/2 in the hippocampal CA3 sector but not in the CA1 area. Thus, the distribution of phospho-ERK 1/2 coincides with hyperglycemia-recruited damage structures. The results suggest that hyperglycemia may influence the outcome of an ischemic insult by modulating signal transduction pathways involving ERK 1/2. © 2001 Academic Press Key Words: hyperglycemia; cerebral ischemia; ERK; MAP kinase; immunocytochemistry.

INTRODUCTION

Recent studies show that hyperglycemia/acidosis suppress the expression of the immediate early gene c-fos (Combs et al., 1992) and of brain-derived neurotrophic factor (BDNF) (Uchino et al., 1997), which is observed as a results of the ischemic transient. It has also been reported that hyperglycemia increases the expression of early-response genes and suppresses late-response genes, which are induced by spreading depression (Koistinaho et al., 1999). Thus, it is likely that glucose may mediate its adverse effects by altering various signal transduction pathways. The response of cells to extracellular stimuli is mediated by a number of intracellular kinases and phosphatases. The members of the mitogen-activated protein kinase (MAPK) have been characterized as central components of the signal transduction pathways in regulating cell proliferation and differentiation. Members of MAPK include c-jun NH 2-terminal kinase (JUK), p38, and extracellular signal-regulated kinases

It has been known for several decades that transient cerebral ischemia induces delayed neuronal death in the dorsolateral crescent of caudate putamen, in the hippocampal CA1 sector, in the neocortex, and in the thalamus. If animals are preloaded with glucose to generate hyperglycemia before the induction of ischemia, brain damage is accelerated, additional structures such as the hippocampal CA3 area, the dentate gyrus, the cingulate cortex, and the substantia nigra are recruited, and postischemic fatal seizure develops (for literature and reviews, see Siesjo¨ et al., 1996; Li & Siesjo¨, 1997). Since hyperglycemia-exaggerated brain damage is incurred over a narrow threshold of plasma glucose concentrations and brain tissue pH values, we have speculated that hyperglycemia-mediated damage is caused by activation of biochemical cascades which have a steep pH dependence (Li et al., 1994, 1995). 0969-9961/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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128 (ERK); the latter being composed of ERK1 (p44) and ERK2 (p42). Various members of the MAPK family are activated by stress signals such as inflammation, cytokines, heat shock protein, ultraviolet light, and ischemia (for literature and reviews, see Seger & Krebs, 1995; Paul et al., 1997). Phosphorylation on both threonine and tyrosine residues is required for full activation of MAPK. Activation of JUK and p38 lead to apoptosis and cell proliferation (Westwick et al., 1995a, 1995b; Verheij et al., 1996; Paul et al., 1997). Activation of ERK 1/2 has been shown either ameliorating or exaggerating cell death (Alessandrini et al., 1999; Hetman et al., 1999). Interestingly, in rat glomeruli and mesangial cells high glucose levels or diabetes are able to induce phosphorylation of ERK 1/2, through activation of protein kinase C (PKC) or cytosolic phospholipase A 2 (cPLA 2) (Haneda et al., 1997). It has not been studied whether hyperglycemia activates phosphorylation of ERK 1/2 in brain tissue, whether hyperglycemia changes the pattern and spatial distribution of phospho-ERK 1/2 induced under normoglycemic ischemia condition, and whether the activation of ERK 1/2 by hyperglycemia is correlated to cell death or survival. To clarify these issues, phosphorylation of ERK 1/2 was studied in ischemia rats subjected to 15 min of forebrain ischemia, and 0.5, 1, and 3 h of reperfusion under both hyperglycemic and normoglycemic conditions by double-staining laser-scanning confocal microscopy and by Western blot analysis. The results suggest a correlation between ERK upregulation and neuronal death in hyperglycemic subjects.

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tained at 37°C with a heating lamp and pad. In the hyperglycemic series, all animals were infused with a 25% glucose solution for 30 min before the induction of ischemia to yield a glucose concentration around 20 mM. Normoglycemic animals were infused with the same amount of 0.9% saline for 30 min. Forebrain ischemia was induced by bilateral clamping of the carotid arterial plus hypotension to 45–50 mmHg by withdrawing and infusing blood through a jugular catheter (Smith et al., 1984). At the end of 15-min ischemia, the clamps were removed and the blood reinfused through the jugular vein. Ventilation was adjusted to yield a mean PaCO 2 of 35– 45 mmHg, a PaO 2 of 100 mmHg, and an arterial pH of 7.35–7.45. Rats destined for 30-min reperfusion were maintained under anesthesia for the entire postischemic period, after which time the brain was collected. For longer reperfusion times, halothane was discontinued at the end of ischemia and all wounds were sutured. The animals were then reanesthetized, tracheotomized, and artificially ventilated to allow collection of brains after 1 or 3 h of reperfusion. Tissue samples for Western studies were obtained by freezing the brains in situ with liquid nitrogen while respiration was maintained with a respirator. Cingulate cortex and dentate gyrus were dissected out in a glove box at a temperature ⫺20°C. For immunostaining, the brain were perfused with ice-cold 4% phosphate-buffered paraformaldehyde while the animal’s respiration was maintained with the respirator. The brains were sectioned with a Vibratome at a thickness of 50 ␮m. Sections were preserved in antifreeze solution until examination of immunostaining.

EXPERIMENTAL PROCEDURES Immunocytochemistry Animal Operation and Ischemia Model Male Wistar rats (Simonsen Laboratory, Gilrey, CA), weighing 280 –320 g, were used in the experiments. Rats were fasted over night prior to operation with free access to tap water. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Ethics Committee. Anesthesia was induced with 3% halothane followed by maintenance with 1–1.5% halothane in an oxygen/nitrous oxide (30/70%) gas mixture. Catheters were inserted into the tail artery and tail vein to allow blood sampling, monitoring of arterial blood pressure, and infusion of glucose. Both common carotid arteries were separated and encircled by loose ligatures. Brain and body temperatures were mainCopyright © 2001 by Academic Press All rights of reproduction in any form reserved.

Double-label fluorescence immunocytochemistry was performed on coronal brain sections (50 ␮m) from both normoglycemic and hyperglycemic sham-operated controls and animals subjected to 15 min of ischemia followed by 0.5, 1, and 3 h of reperfusion. A monoclonal antibody against phospho-ERK 1/2 was obtained from New England Biolabs (Beverly, MA). The brain sections were washed twice in phosphatebuffered saline (PBS) for 5 min at room temperature (RT) and then washed in PBS containing 0.2% Triton X-100 (TX-100) twice, 10 min each. The sections in PBS/0.2% TX-100 solution was briefly heated in a microwave oven before nonspecific binding sites were blocked in 3% bovine serum albumin (BSA) in PBS/0.2% TX-100 for 30 min. The primary antibody was diluted in PBS/0.1% TX-100 and 3% BSA at 1:500 concentration.

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After overnight incubation at 4°C in the primary antibody, the sections were washed in PBS/0.1% TX-100 three times 10 min each at RT. The sections were then incubated with the secondary antibody FITC-conjugated affinipure donkey anti-mouse IgG 1:200 and propidium iodide (1:300) in TBS containing 0.1% Triton X-100 and 3% BSA for 2 h at 4°C. Sections were washed several times in PBS/0.1% TX-100, and then mounted on glass slides and coverslipped using Gelovatol. The slides were analyzed on a Bio-Rad MRC1024 laser-scanning confocal microscope. Preparation of Nuclear Extracts and Subcellular Fractions In both normoglycemic and hyperglycemic animals, subcellular fractions were prepared from sham-operated animals and from ischemic brains after 0.5, 1, and 3 h of reperfusion. Samples were prepared at 4°C from each of four animals per condition. Brain tissues in the cingulate cortex and dentate gyrus were homogenized using a Dounce homogenizer (30 strokes) in 15 times the volume of homogenization buffer containing 15 mM Tris base/HCl, pH 7.6, 1 mM DTT, 0.25 M sucrose, 1 mM MgCl 2, 1.25 ␮g/ml pepstatin A, 10 ␮g/ml leupeptin, 2.5 ␮g/ml aproptonin, 0.5 mM PMSF, 2.5 mM EDTA, 1 mM EGTA, 0.1 M Na 3 VO 4, 50 mM NaF, and 2 mM sodium pyrophosphate. The homogenates were then centrifuged at 700 rcf at 4°C for 10 min. The resultant pellet (P1) was used as the crude nuclear fraction. The supernatants were further centrifuged at 10,000 rcf for 10 min to a obtain crude synaptosomal fraction (P2) and the resulting supernatants were further centrifuged at 75,000 rcf for 1 h at 4°C to separate cytosolic fraction (S3) and microsomal fraction (P3). P1, P2, and P3 were suspended with homogenization buffer containing 0.1% TX-100. The samples were kept at ⫺80°C until analyses. The protein concentration of the samples was determined by a Lowry kit. Western Blot Analysis Western blot analysis was carried out with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) according to the method of Laemmli. One sample containing 40 ␮g protein was applied to each line in a slab gel of SDS–PAGE. Following electrophoresis, proteins were transferred to an immobilon-P membrane. The membrane was incubated with primary antibody against phospho-ERK 1/2 (New England Biolabs) at a dilution of 1:3000 for overnight at 4°C and then incubated with horseradish

peroxidase-conjugated anti-mouse secondary antibody at 1:3000 dilution (Amersham Life Science) for 1 h at room temperature. The blots were developed using the enhanced chemoluminescence (ECL) detection method (Amersham). Each protein band on the western blot was derived from one animal. Two samples in each group were run on the same gel and analyzed at the same time. Each group consisted of four animals. Quantification of Brain Damage Vibratome sections were stained with a combination of acid fuchsin/celestine blue. Bright red-stained acidophilic neurons with a shrunken, triangularshaped, and dense purple nuclei were considered dead neurons. A five-point scale was used, where grade 0 denotes no observed damage, grade 1, a few scattered dead neurons per section, grade 2, 1–9% of damaged neurons, grade 3, 10 –50%, and grade 4, ⬎50%. Statistics Relative density of ERK1 and ERK2 was measured with a Kodak image program. Comparison of recirculation groups with sham-operated controls in normoglycemic and hyperglycemic animals was made by ANOVA followed by Scheffe’s test. Comparison between normoglycemic and hyperglycemic animals at the identical recirculation time point was made by unpaired Student t test. A P value less than 0.05 was regarded as statistically significant. All tests were twosided.

RESULTS Physiological Parameters Mean blood glucose concentration was 4 –5 and 20 –24 mmol/L in normoglycemic and hyperglycemic rats, respectively. Physiological parameters were well controlled. PaCO 2 was maintained close to 35– 44 mmHg, PaO 2 to 100 –130 mmHg, arterial pH to 7.40 – 7.46, and blood pressure to 100 –113 mmHg. Both core and head temperatures were controlled at 36.9 –37.4°C. There were no significant differences between the normoglycemic and hyperglycemic groups. Brain Damage As remarked, brain sections were stained with a combination of acid fuchsin/celestine blue and were Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1. Double immunostaining confocal images for phospho-ERK (green) and propidium iodide (red) in cingulate cortex and dentate gyrus in both normoglycemic (NG) and hyperglycemic (HG) rats subjected to sham-operation (c) or to 15-min 2VO followed by 0.5, 1, and 3 h of reperfusion. Transient increases of phospho-ERK were observed after 0.5 h of recovery in NG animals in both cingulate cortex and dentate gyrus. More marked and persistent induction of phospho-ERK was presented in HG animals in both structures.

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Distribution of Phospho-ERK 1/2

FIG. 2. Double immunostaining confocal images showing different induction of phospho-ERK in the hippocampal CA1 and CA3 regions in NG (a– c) and HG (d–f) animals after 0.5 h of recovery. Phospho-ERK is not increased in NG animals in both CA1 (a, b) and CA3 (a, c) areas. In contrast, Phospho-ERK is highly increased in HG animals in the CA3 (d, f), but not in the CA1 subregions (d, e).

double-blind examined with light microscopy at 200⫻ and 400⫻ magnifications by direct visual counting of dead neurons. Neuronal death was not observed in the hippocampal CA1, CA3, or dentate gyrus subregions, nor was it found in neocortical structures up to 3 h after recirculation in normoglycemic animals. In hyperglycemic animals, damage to the hippocampal CA1, CA3, and dentate gyrus regions was not observed after 0.5 and 1 h of recovery; however, grade 1 damage was observed in dentate gyrus after 3 h of reperfusion in three rats and grade 3 damage was observed in the CA3 area in two rats. In neocortical areas, a few scattered dead neurons (grade 1) were observed in rats after 0.5 h of recovery, and grade 2 and 3 damage in five rats after 1 h of recovery. At 3 h of recirculation, all six rats had neocortical damage, with one rat showing grade 1, one grade 2, and four grade 4 damage.

Brain sections from sham-operated controls (C) and those subjected to 15 min ischemia followed by 0.5, 1, and 3 h of reperfusion in both normoglycemic and hyperglycemic animals were double-stained with a monoclonal antibody against phospho-ERK 1/2 and with propidium iodide to label nucleic acids. The sections were examined by laser-scanning confocal microscopy. In Figs. 1 and 2, the red color represents nucleic acids stained by propidium iodide, while the green color represents fluorescein-conjugated antibodies specific for phospho-ERK 1/2. When the red and the green are overlaid the color turns yellow. In the cingulate cortex of normoglycemic animals, phosphoERK 1/2 was slightly and transiently upregulated after 0.5 h of recovery and returned to control level after 1 and 3 h (Fig. 1, Cingulate cortex, upper panel). Induction of phospho-ERK 1/2 was dramatically increased in hyperglycemic animals after 0.5 h of recovery and the upregulation was sustained after 1 and 3 h of recovery (Fig. 1, Cingulate cortex, lower panel). Changes of phospho-ERK 1/2 in the frontoparietal cortex were similar to those observed in the cingulate cortex (data not shown). Thus, induction of phosphoERK 1/2 was not observed in normoglycemic rats but it was increased in hyperglycemic ones with a peak increase after 1 h of recovery. ERK 1/2 was transiently phosphorylated after 0.5 h of recovery in dentate gyrus in normoglycemic animals and dephosphorylated thereafter (Fig. 1, Dentate gyrus, upper panel). The induction of ERK 1/2 after 0.5 h recovery in normoglycemic group appears higher than that in hyperglycemic group. The phosphorylation was also induced after 0.5 h of recovery in hyperglycemic rats; however, unlike normoglycemic animals, the hyperglycemic ones showed continued upregulation after 1 and 3 h of recovery (Fig. 1, Dentate gyrus, lower panel). No obvious phosphorylation of ERK 1/2 was observed in the hippocampal CA1 sector in either normoglycemic or hyperglycemic animals after 0.5 h of recovery (Figs. 2a, 2b, and 2d, 2e); in contrast, there was a clear difference for the expression of phospho-ERK 1/2 in the hippocampal CA3 sector between normoglycemic and hyperglycemic animals (Figs. 2a and 2d). Thus, although there was no induction of phospho-ERK 1/2 in normoglycemic rats after 0.5 h of recovery (Figs. 2a–2c), the induction of phospho-ERK 1/2 was robustly increased in hyperglycemic rats after 0.5 h of recovery (Figs. 2d and 2f). The upregulation of ERK 1/2 persisted after 1 h and then decreased after 3 h of recovery (data not shown). Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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FIG. 3. Upper panel shows Western blot analysis of phospho-ERK 1/2 in cytosolic fraction from the cingulate cortical tissue after ischemia. Each lane represents one rat. Phosphorylation of ERK 1/2 is transiently increased after 0.5 h of recovery in normoglycemic animals. Compared to normoglycemic rats, activation of ERK is markedly increased after 0.5, 1, and 3 h of recovery in hyperglycemic animals. Lower panels are semiquantitative changes of phospho-ERK 1/2 after ischemia in normo- and hyperglycemic animals. Data are mean ⫾ SD (n ⫽ 4 per time point). *P ⬍ 0.05 versus control (ANOVA followed by Scheffe’s test); †P ⬍ 0.05 versus NG at same recovery time (unpaired t test).

Western blot analyses of phospho-ERK 1/2 were performed in cytosolic fractions collected from the cingulate cortex and the dentate gyrus. Consistent with the findings observed by confocal microscopy, Western blots revealed a transient mild increase of active ERK-42 and -44 kDa after 0.5 h recovery in the cingulate cortex in normoglycemic animals and a marked increase after 0.5, 1, and 3 h of recovery in hyperglycemic animals (Fig. 3, upper panel). Semiquantitative analysis of the Western blots showed a more significant increase in hyperglycemic than in normoglycemic animals (Fig. 3, lower panel). ERK 1/2 were upregulated in the dentate gyrus after 0.5 h of recovery and then declined to control level after 1 and 3 h of recovery in normoglycemic animals (Fig. 4, upper panel). Mild upregulation of phospho ERK 1/2 was induced after 0.5 h of recovery and there was a more marked upregulation after 1 and 3 hrs of recovery in hyperglycemic animals (Fig. 4, upper panel). Relative density was given in Fig. 4, lower panel.

DISCUSSION Spatial distribution and temporal changes of phospho-ERK 1/2 were investigated using a specific antiCopyright © 2001 by Academic Press All rights of reproduction in any form reserved.

body in both normoglycemic and hyperglycemic rats subjected to sham operation or to 15 min of transient forebrain ischemia followed by 0.5, 1, and 3 h of reperfusion. The results obtained from hyperglycemic animals were compared with glucose-infused shamoperated controls and saline-infused normoglycemic ischemic animals as well. Transient phosphorylation of ERK 1/2 was induced by ischemia in normoglycemic animals after 0.5 h of reperfusion in dentate gyrus and cingulate cortex, but not in CA1, CA3, and parietal cortical regions. These results are identical to those published by Hu et al. (2000) who showed upregulation of ERK in the dentate gyrus but not in the CA1 sector. Compared to normoglycemic animals, hyperglycemia induced more marked and persistent activation of ERK 1/2 in the cingulate and parietal cortex, and CA3 regions, but not in the CA1 region. In the dentate gyrus, phosphorylation of ERK 1/2 was weaker in hyperglycemia at 30 min but stronger at 1 and 3 h of reperfusion than in normoglycemia. This may reflect a biphasic effect of hyperglycemia on cell death. Thus, hyperglycemia initially promotes mitochondrial respiration and preserves mitochondrial energetic status in the early reperfusion stage (Hillered et al., 1985; Wagner & Myers, 1986) but later accelerates

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FIG. 4. Upper panel shows Western blot analysis of phospho-ERK 1/2 in cytosolic fraction from the dentate gyrus after ischemia. Phosphorylation of ERK 1/2 are transiently increased after 0.5 h of recovery in normoglycemic animals. A delayed increase of phospho-ERK 1/2 is observed after 1 and 3 h of recovery in hyperglycemic animals. Each lane represents one rat. Data are mean ⫾ SD (n ⫽ 4 per time point). *P ⬍ 0.05 versus control (ANOVA followed by Scheffe’s test); †P ⬍ 0.05 versus NG at same recovery time (unpaired t test).

cell death through undefined pathways (Smith et al., 1988; Li et al., 1999). It is unlikely that the hyperglycemia-accelerated injury in the reperfusion phase is due to lactic acid buildup since intra- and extracellular pH returned to normal level after 15–30 min of recirculation in both normo- and hyperglycemic animals (Smith et al., 1986; Li et al., 1995). As mentioned in the introduction, it is not known whether activation of MAPK by phosphorylation is detrimental or beneficial for the cell. Results from Hetman et al. (1999) showed that ERK is activated in cortical neurons during camptothecin-induced apoptosis, and that inhibition of ERK increases apoptosis. In a recent article, Hu et al. (2000) showed that the ERK pathway was activated in the neuronal population, which will survive after transient cerebral ischemia and reperfusion injury. It has also shown that ischemic preconditioning induces phosphorylation of ERK in the rat and gerbil hippocampal CA1 neurons (Shamloo et al., 1999; Sugino et al., 2000). In the present experiments, phosphorylation of ERK was induced in the areas where neurons are destined to die in hyperglycemic animals. Thus, it appears that hyperglycemia-induced ERK upregulation may be related to neuronal death. This contention was supported by studies

showing that the ERK pathway is activated during focal or global cerebral ischemia, and that inhibition of this pathway reduces infarct volume (Campos-Gonzalez & Kindy, 1992; Alessandrini et al., 1999). Increase of tyrosine phosphorylation was prevented by the N-methyl-d-aspartate (NMDA) receptor blocker MK-801 and calcium channel blockers (CamposGonzalez & Kindy, 1992). Tyrosine phosphorylation was also prevented under hypothermic condition. These results suggest that MAP kinase may mediate neuronal death after ischemia and reperfusion injury. However, it is also possible that the activation of ERK is a survival response that is not sufficient to protect the cell from the neurotoxic insults. It is not clear by which pathway hyperglycemia activates ERK 1/2. ERK 1/2 are activated by Rasdependent signal transduction pathways through mitogen-activated, ERK-activating kinase (MEK) or by protein kinase C (PKC) (Seger and Krebs, 1995). Glucose has been shown to increase the activation of PKC in rat glomeruli and glomerular mesangial cells (Ayo et al., 1991; Haneda et al., 1997), aortic smooth muscle cells (Igarashi et al., 1999), and brain tissue (Bhardwaj et al., 1999). Thus it is likely that hyperglycemia activates MAPK through activation of PKC. MAPK was Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

134 found, on the one hand, to phosphorylate and activate Elk-1, one of the ternary complex factors. The activation of Elk-1 will result in expression of c-fos and c-jun, which relate to cell proliferation. On the other hand, MAPK was also found to phosphorylate and activate cPLA 2. Activation of cPLA 2 may in turn lead to the enhancement of arachidonic acid release. The products of the PLA 2-catalyzed reaction are further metabolized to eicosanoids, platelet-activating factor, and lysophosphatidic acid. All of these are recognized as bioactive lipids that can cause loss of essential membrane glycerophospholipids, resulting in altered membrane permeability, ion homeostasis, increased free fatty acid release, and accumulation of lipid peroxides, and eventually neurodegeneration or apoptosis (for reviews, see Farooqui et al., 1997; Farooqui & Horrocks, 1998). In summary, hyperglycemia markedly increased phosphorylation of ERK 1/2 in the cingulate cortex, frontoparietal cortex, hippocampal CA3, and dentate gyrus areas, structures where exaggerated ischemic brain damage is induced by hyperglycemia. Hyperglycemia may activate ERK 1/2 through PKC and activated ERK 1/2 may further stimulate cPLA 2, leading to lipid peroxidation and oxidative damage to membrane proteins and eventually cell death. Further experiments with treatment of MAPK inhibitors in animals subjected to cerebral ischemia in hyperglycemic condition is highly justified to define the role of MAPK in hyperglycemia-enhanced ischemic brain damage.

ACKNOWLEDGMENTS This work was supported by National Institute of Health Grants NS07838 and NS36810, Hawaii Community Foundation, and Queen Emma Research Foundation.

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