Apoptotic Changes in the Aged Brain Are Triggered by Interleukin-1β-induced Activation of p38 and Reversed by Treatment with Eicosapentaenoic Acid
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m205289200
ISSN1083-351X
AutoresDarren S.D. Martin, Peter E. Lonergan, Barry Boland, Marie P. Fogarty, Marcella Brady, David F. Horrobin, Veronica A. Campbell, Marina A. Lynch,
Tópico(s)Adipose Tissue and Metabolism
ResumoAmong the several changes that occur in the aged brain is an increase in the concentration of the proinflammatory cytokine interleukin-1β that is coupled with a deterioration in cell function. This study investigated the possibility that treatment with the polyunsaturated fatty acid eicosapentaenoic acid might prevent interleukin-1β-induced deterioration in neuronal function. Assessment of four markers of apoptotic cell death, cytochrome c translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and terminal dUTP nick-end staining, revealed an age-related increase in each of these measures, and the evidence presented indicates that treatment of aged rats with eicosapentaenoate reversed these changes as well as the accompanying increases in interleukin-1β concentration and p38 activation. The data are consistent with the idea that activation of p38 plays a significant role in inducing the changes described since interleukin-1β-induced activation of cytochrome ctranslocation and caspase-3 activation in cortical tissue in vitro were reversed by the p38 inhibitor SB203580. The age-related increases in interleukin-1β concentration and p38 activation in cortex were mirrored by similar changes in hippocampus. These changes were coupled with an age-related deficit in long term potentiation in perforant path-granule cell synapses, while eicosapentaenoate treatment was associated with reversal of age-related changes in interleukin-1β and p38 and with restoration of long term potentiation. Among the several changes that occur in the aged brain is an increase in the concentration of the proinflammatory cytokine interleukin-1β that is coupled with a deterioration in cell function. This study investigated the possibility that treatment with the polyunsaturated fatty acid eicosapentaenoic acid might prevent interleukin-1β-induced deterioration in neuronal function. Assessment of four markers of apoptotic cell death, cytochrome c translocation, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and terminal dUTP nick-end staining, revealed an age-related increase in each of these measures, and the evidence presented indicates that treatment of aged rats with eicosapentaenoate reversed these changes as well as the accompanying increases in interleukin-1β concentration and p38 activation. The data are consistent with the idea that activation of p38 plays a significant role in inducing the changes described since interleukin-1β-induced activation of cytochrome ctranslocation and caspase-3 activation in cortical tissue in vitro were reversed by the p38 inhibitor SB203580. The age-related increases in interleukin-1β concentration and p38 activation in cortex were mirrored by similar changes in hippocampus. These changes were coupled with an age-related deficit in long term potentiation in perforant path-granule cell synapses, while eicosapentaenoate treatment was associated with reversal of age-related changes in interleukin-1β and p38 and with restoration of long term potentiation. interleukin-1β poly(ADP-ribose) polymerase terminal dUTP nick-end labeling long term potentiation type I IL-1 receptor IL-1R antagonist eicosapentaenoic acid phosphate-buffered saline 7-amino-4-trifluoromethyl coumarin analysis of variance Increased expression of the proinflammatory cytokine interleukin-1β (IL-1β)1has been linked with neurodegenerative disorders like Down's syndrome, Alzheimer's disease, and Parkinson's disease (1Griffin W.S.T. Stanley L.C. Ling C. White L. MacLeod V. Perrot L.J. White III, C.L. Araoz C. Proc. Natl. Acad. Sci. U. S. 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Rev. 2001; 21: 129-145Crossref PubMed Scopus (247) Google Scholar) has been closely linked with apoptotic cell death. Significantly, an increase in p38 activity has been coupled with apoptotic changes in Alzheimer's disease (17Zhu X. Rottkamp C.A. Boux H. Takeda A. Perry G. Smith M.A. J. Neuropathol. Exp. Neurol. 2000; 59: 880-888Crossref PubMed Scopus (305) Google Scholar, 18Hensley K. Floyd R.A. Zheng N.Y. Nael R. Robinson K.A. Nguyen X. Pye Q.N. Stewart C.A. Geddes J. Markesbery W.R. Patel E. Johnson G.V. Bing G. J. Neurochem. 1999; 72: 2053-2058Crossref PubMed Scopus (316) Google Scholar). Concomitant increases in IL-1β concentration and p38 activity have been reported in the aged rat brain (19Murray C.A. Lynch M.A. J. Neurosci. 1998; 18: 2974-2981Crossref PubMed Google Scholar, 20Murray C.A. Lynch M.A. J. Biol. Chem. 1998; 273: 12161-12168Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 21O'Donnell E. Vereker E. Lynch M.A. Eur. J. Neurosci. 2000; 12: 345-352Crossref PubMed Scopus (115) Google Scholar); in hippocampus these changes are correlated with compromised synaptic function and with an age-related impairment in long term potentiation (LTP) (19Murray C.A. Lynch M.A. J. Neurosci. 1998; 18: 2974-2981Crossref PubMed Google Scholar, 20Murray C.A. Lynch M.A. J. Biol. Chem. 1998; 273: 12161-12168Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 21O'Donnell E. Vereker E. Lynch M.A. Eur. J. Neurosci. 2000; 12: 345-352Crossref PubMed Scopus (115) Google Scholar, 22McGahon B.M. Martin D.S.D. Horrobin D.F. Lynch M.A. Neuroscience. 1999; 94: 305-314Crossref PubMed Scopus (205) Google Scholar), while consistent with the high expression of IL-1β and IL-1RI in hippocampus is the finding that the cytokine depresses LTP in dentate gyrus (8Vereker E. O'Donnell E. Lynch M.A. J. Neurosci. 2000; 20: 6811-6819Crossref PubMed Google Scholar, 19Murray C.A. Lynch M.A. J. Neurosci. 1998; 18: 2974-2981Crossref PubMed Google Scholar, 20Murray C.A. Lynch M.A. J. Biol. Chem. 1998; 273: 12161-12168Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar,23Cunningham A.J. Murray C.A. O'Neill L.A.J. Lynch M.A. O'Connor J.J. Neurosci. Lett. 1996; 203: 1-4Crossref PubMed Scopus (348) Google Scholar, 24Lynch M.A. Prog. Neurobiol. 1998; 56: 1-19Crossref PubMed Scopus (144) Google Scholar). Significantly, we have recently reported that the age-related increases in IL-1β concentration and c-Jun NH2-terminal kinase activity, as well as the decrease in LTP, are reversed by treatment with the n-3 polyunsaturated fatty acid docosahexaenoic acid (22McGahon B.M. Martin D.S.D. Horrobin D.F. Lynch M.A. Neuroscience. 1999; 94: 305-314Crossref PubMed Scopus (205) Google Scholar). In this study we have attempted to identify the downstream consequences of the coupled age-related increases in IL-1β concentration and p38 activation in neuronal tissue. In particular, we have focused on assessing whether these changes might trigger apoptotic changes in neuronal tissue as it does in other tissues and have analyzed the effect of the ethyl ester of the ω-3 fatty acid eicosapentaenoic acid (EPA) on age-related changes in cortex and hippocampus. The data indicate that dietary manipulation reversed several changes in the aged cortex that are indicative of apoptotic cell death as well as age-related changes in IL-1β concentration, p38 activation, and LTP in hippocampus. Groups of young and aged male Wistar rats (300–350 g), maintained at an ambient temperature of 22–23 °C under a 12-h light-dark schedule, were subdivided into those that were fed on a diet enriched in eicosapentaenoic acid (ethyl eicosapentaenoate, 10 mg/rat/day for 3 weeks and 20 mg/rat/day for 5 weeks; Laxdale Research Ltd.) or standard laboratory chow for 8 weeks. Daily food intake was assessed for 2 weeks prior to commencement of the treatment: mean values (±S.E.) were 21.25 ± 1.4 and 18.55 ± 0.6 g/day for 4- and 22-month-old rats, respectively. At this time the mean body weights of young rats assigned to control and experimental groups were 265.6 ± 9.1 and 250.2 ± 11.7 g, respectively; corresponding values in aged rats were 483.6 ± 9.8 and 481.2 ± 7.9 g, respectively. Diet was prepared fresh each day, and rats were offered 100% of their daily intake. Mean daily food intake in all groups remained unchanged throughout the 8-week treatment period, and at the end of this time mean body weights of young rats assigned to control and experimental groups were 366.4 ± 13.4 and 354.5 ± 19.4 g, respectively; corresponding values in aged rats were 473.7 ± 11.4 and 462.9 ± 6.3 g, respectively. At this time rats were 4 and 22 months old. Rats were maintained under veterinary supervision for the duration of this experiment. At the end of the 8-week treatment, LTP was induced as described previously (19Murray C.A. Lynch M.A. J. Neurosci. 1998; 18: 2974-2981Crossref PubMed Google Scholar). Rats were anesthetized by intraperitoneal injection of urethane (1.5g/kg), recording and stimulating electrodes were placed in the molecular layer of the dentate gyrus (2.5 mm lateral and 3.9 mm posterior to bregma) and perforant path, respectively (angular bundle, 4.4 mm lateral to lambda), and stable baseline recordings were made before electrophysiological recording at test shock frequency (1/30 s) commenced for 10 min before and 40 min after tetanic stimulation (three trains of stimuli; 250 Hz for 200 ms; intertrain interval, 30 s). Rats were then killed by decapitation, and the hippocampus and cortex were removed, cross-chopped into slices (350 × 350 μm), and frozen separately in 1 ml of Krebs' solution (136 mm NaCl, 2.54 mm KCl, 1.18 mm KH2PO4, 1.18 mmMgSO4·7H2O, 16 mmNaHCO3, 10 mm glucose, 1.13 mmCaCl2) containing 10% dimethyl sulfoxide (22McGahon B.M. Martin D.S.D. Horrobin D.F. Lynch M.A. Neuroscience. 1999; 94: 305-314Crossref PubMed Scopus (205) Google Scholar). For analysis, thawed slices were rinsed three times in fresh buffer and used as described below. IL-1β concentration was analyzed in homogenate prepared from cortex and hippocampus by enzyme-linked immunosorbent assay (R&D Systems). Antibody-coated (100 μl; 1.0 μg/ml final concentration diluted in phosphate-buffered saline (PBS), pH 7.3; goat anti-rat IL-1β antibody) 96-well plates were incubated overnight at room temperature, washed several times with PBS containing 0.05% Tween 20, blocked for 1 h at room temperature with 300 μl of blocking buffer (PBS, pH 7.3 containing 5% sucrose, 1% bovine serum albumin, and 0.05% NaN3), and washed. IL-1β standards (100 μl; 0–1,000 pg/ml in PBS containing 1% bovine serum albumin) or samples (homogenized in Krebs' solution containing 2 mm CaCl2) were added, and incubation proceeded for 2 h at room temperature. Secondary antibody (100 μl; final concentration, 350 ng/ml in PBS containing 1% bovine serum albumin and 2% normal goat serum; biotinylated goat anti-rat IL-1β antibody) was added and incubated for 2 h at room temperature. Wells were washed, detection agent (100 μl; horseradish peroxidase-conjugated streptavidin; 1:200 dilution in PBS containing 1% bovine serum albumin) was added, and incubation continued for 20 min at room temperature. Substrate solution (100 μl; 1:1 mixture of H2O2 and tetramethylbenzidine) was added and incubated at room temperature in the dark for 1 h after which time the reaction was stopped using 50 μl of 1 mH2SO4. Absorbance was read at 450 nm, and values were corrected for protein (25Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214435) Google Scholar) and expressed as pg of IL-1β/mg of protein. p38 phosphorylation was analyzed in samples of homogenate prepared from hippocampus and cortex; cytosolic cytochromec and expression of 116-kDa PARP were analyzed in cortical tissue. p38 activity was also assessed in freshly prepared hippocampal and cortical tissue that was incubated for 20 min in the absence or presence of IL-1β (3.5 ng/ml), while cytochrome ctranslocation was assessed in vitro following incubation of slices of cortex with IL-1β (3.5 ng/ml) in the presence or absence of IL-1ra (350 ng/ml) or SB203580 (50 μm). For analysis of p38 in all experiments and also for PARP, homogenate was diluted to equalize for protein concentration, and aliquots (10 μl, 1 mg/ml) were added to 10 μl of sample buffer (0.5 mm Tris-HCl, pH 6.8, 10% glycerol, 10% SDS, 5% β-mercaptoethanol, 0.05% (w/v) bromphenol blue), boiled for 5 min, and loaded onto 10% SDS gels. In the case of cytochrome c, cytosolic fractions were prepared by homogenizing slices of cortex in lysis buffer (20 mm hEPES, pH 7.4, 10 mm KCl, 1.5 mmMgCl2, 1 mm EGTA, 1 mm EDTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, 5 μg/ml pepstatin A, 2 μg/ml leupeptin, 2 μg/ml aprotinin), incubating for 20 min on ice, and centrifuging (15,000 × g for 10 min at 4 °C). The supernatant (i.e. cytosolic fraction) was suspended in sample buffer (150 mm Tris-HCl, pH 6.8, 10% (v/v) glycerol, 4% (w/v) SDS, 5% (v/v) β-mercaptoethanol, 0.002% (w/v) bromphenol blue) to a final concentration of 300 μg/ml, boiled for 3 min, and loaded (6 μg/lane) onto 12% gels. In all cases proteins were separated by application of 30 mA constant current for 25–30 min, transferred onto nitrocellulose strips (225 mA for 75 min), and immunoblotted with the appropriate primary and secondary antibodies. In the case of p38, anti-phospho-p38 (Santa Cruz Biotechnology; 1:500 in phosphate-buffered saline-Tween (0.1% Tween 20) containing 2% nonfat dried milk) and peroxidase-linked anti-mouse IgM (1:1,000; Amersham Biosciences) were used. In the case of PARP, we immunoblotted with an antibody (1:2,000) raised against the epitope corresponding to amino acids 764–1014 of poly(ADP-ribose) polymerase of human origin (Santa Cruz Biotechnology), and immunoreactive bands were detected using peroxidase-conjugated anti-rabbit IgG (Sigma) and ECL (Amersham Biosciences). To assess cytochrome c, a rabbit polyclonal antibody raised against recombinant protein corresponding to amino acids 1–104 of cytochromec (Santa Cruz Biotechnology) was used. In addition to loading equal amounts of protein, some blots were reprobed for analysis of total (rather than phosphorylated) p38, and in other cases blots were probed with an anti-actin antibody to confirm equal loading. Data from these experiments showed no differences between treatment groups. In all experiments, immunoreactive bands were detected using peroxidase-conjugated anti-rabbit antibody (Sigma) and ECL (AmershamBiosciences). Quantification of protein bands was achieved by densitometric analysis using two software packages, Grab It (Grab It Annotating Grabber, Version 2.04.7, Synotics, UVP Ltd.) and Gelworks (Gelworks ID, Version 2.51, UVP Ltd.) for photography and densitometry, respectively. Slices of cortical tissue prepared from young and aged rats were washed, homogenized in lysis buffer (400 μl; 25 mm HEPES, 5 mmMgCl2, 5 mm dithiothreitol, 5 mmEDTA, 2 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin, pH 7.4), incubated on ice for 20 min, analyzed for protein concentration, and diluted to equalize for protein concentration. In some experiments, slices prepared from control young rats were incubated for 60 min at 37 °C in the presence or absence of IL-1β (3.5 ng/ml) to which IL-1ra (350 ng/ml) or SB203580 (50 μm) was added; these samples were treated as described above. All samples (98 μl) were added to 2 μl of caspase-3 substrate (Ac-DEVD-AFC peptide, Alexis Corp.; 5 μm), transferred to a 96-well plate, and incubated for 1 h at 37 °C. Fluorescence was assessed (excitation, 390 nm; emission, 510 nm), and enzyme activity was calculated with reference to a standard curve of AFC (0–10 μm) concentration versusabsorbance. Total RNA was extracted from cortical neurons (26MacManus A. Ramsden M. Murray M. Pearson H.A. Campbell V. J. Biol. Chem. 2000; 275: 4713-4718Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) using TRI reagent (Sigma). cDNA synthesis was performed on 1 μg of total RNA using oligo(dT) primer (Superscript reverse transcriptase, Invitrogen). Equal amounts of cDNA were used for PCR amplification for a total of 30 cycles at 94 °C for 1 min and 58 °C for 2 min. A final extension step was carried out at 70 °C for 10 min. Multiplex PCR was performed using the Quantitative PCR Cytopress Detection kit (Rat Apoptosis Set 2; BioSource International, Camarillo, CA) generating caspase-3 PCR products of 320 bp and glyceraldehyde-3-phosphate dehydrogenase PCR products of 532 bp. The PCR products were analyzed by electrophoresis on 2% agarose gels, photographed, and quantified using densitometry. Expression of glyceraldehyde-3-phosphate dehydrogenase mRNA was used as a standard to quantify the relative expression of caspase-3 mRNA. Cortical slices (350 × 350 μm) were incubated at 37 °C for 30 min in HEPES-buffered Krebs' solution (145 mm NaCl, 5 mm KCl, 1 mmMgCl2, 2 mm CaCl2, 1 mmMg2SO4, 1 mmKH2PO4, 10 mm glucose, 30 mm HEPES at pH 7.4) containing trypsin (1 mg/ml), DNase (1,600 kilounits/liter), protease X (1 mg/ml), and protease XIV (1 mg/ml). Slices were washed, triturated, and passed through a nylon mesh filter. Cells were centrifuged (1,000 rpm for 1 min), resuspended in HEPES-buffered Krebs' solution, plated onto coverslips, fixed in 4% paraformaldehyde in PBS (w/v) for 30 min, and permeabilized in 0.1% Triton in PBS (v/v) for 15 min. Cells were incubated in normal goat serum in PBS (v/v) to block nonspecific binding, treated with anti-phosphospecific p38 antibody (1:100; Santa Cruz Biotechnology) or anti-caspase-3 (1:500; BioSource), and incubated overnight at 4 °C. Cells were washed and incubated in the dark for 2 h in either fluorescein isothiocyanate-labeled goat anti-mouse IgG and IgM (1:100; BioSource) or R-phycoerythrin-labeled goat anti-rabbit IgG (1:100; BioSource) to visualize labeling with p38 and caspase-3, respectively. Following a further wash, slides were mounted using 2 mg/ml p-phenylenediamine in 50% glycerol in PBS (v/v) and sealed. Fluorescence was analyzed using the Bio-Rad MRC-1024 laser scanning confocal imaging system in which the fluorochromes were excited by laser light emitted at 565 and 494 nm and detected at 578 and 520 nm, which measured bound R-phycoerythrin and fluorescein isothiocyanate, respectively. Cells were analyzed at ×63 magnification under oil immersion with the laser at 100% power. The images were analyzed using the Bio-Rad software, and the Kalman filter was used to decrease background. In this system R-phycoerythrin-labeled cells are stained red, and fluorescein isothiocyanate-labeled cells are stained green. In a separate series of experiments, groups of aged and young rats were killed and brains were rapidly removed, coated in OCT compound, immersed in an isopentane bath over liquid nitrogen, and used to prepare sections for analysis of phosphorylated p38 as described above. Apoptotic cell death was assessed using the DeadEnd colorimetric apoptosis detection system (Promega). Cells were permeabilized and fixed in paraformaldehyde as described above. In separate experiments cultured cortical neurons were prepared from neonatal rats as described previously (26MacManus A. Ramsden M. Murray M. Pearson H.A. Campbell V. J. Biol. Chem. 2000; 275: 4713-4718Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) and maintained in neurobasal medium for 12 days before incubating in the absence or presence of IL-1β (5 ng/ml) for 72 h. Biotinylated nucleotide was incorporated at 3′-OH DNA ends by incubating cells with terminal deoxynucleotidyltransferase for 30 min at 37 °C. Washed cells were incubated in horseradish peroxidase-labeled streptavidin and then incubated in 3,3′-diaminobenzidine chromogen solution, and TUNEL-positive cells were calculated as a proportion of the total cell number. IL-1β concentration and p38 activity were both significantly increased in cortical tissue prepared from aged rats fed on the control diet compared with young rats (p < 0.05, ANOVA; Fig. 1, a andb), but EPA suppressed these age-related changes so that the values in tissue prepared from EPA-treated rats were not significantly different from control values. In vitro analysis revealed that IL-1β significantly enhanced p38 activity in cortical tissue (Fig. 1c). In parallel with this observation, we found that cytochrome c translocation was significantly increased in cortical tissue prepared from aged rats fed on the control diet compared with tissue prepared from either group of young rats (p < 0.05, ANOVA; Fig.2a). A causal relationship between the age-related changes in cytochrome ctranslocation and IL-1β is suggested by the finding that cytochromec translocation was significantly enhanced by IL-1β (p < 0.05, ANOVA; Fig. 2b) and that this action relied on IL-1RI activation since the IL-1β-induced change was inhibited by IL-1ra. Fig. 2b also demonstrates that the IL-1β-induced change was inhibited by SB203580 suggesting that the effect was mediated by activation of p38.Figure 2The age-related increase in cytochromec translocation is abolished by EPA and mimicked by IL-1β.a, cytochromec translocation was significantly enhanced in cortical tissue prepared from aged rats fed on the control diet compared with young rats fed on control diet (*, p < 0.05, ANOVA; compare lane 3 with lane 1), but this change was not evident in tissue prepared from aged rats fed on the EPA-enriched diet (lane 4). b, incubation of cortical tissue in the presence of IL-1β (lane 2) significantly increased cytochrome c translocation (*, p < 0.05, ANOVA), but this effect was inhibited by IL-1ra (lane 4) and by SB203580 (lane 6); neither IL-1ra (lane 3) nor SB203580 (lane 5) affected cytochrome ctranslocation (n = 6 in each group). Con, control; SB, SB203580.View Large Image Figure ViewerDownload Hi-res image Download (PPT) One downstream consequence of cytochrome c translocation is activation of caspase-3, therefore we analyzed enzyme activity in tissue prepared from aged and young rats fed on either the control or experimental diet. Fig. 3ademonstrates that there was a significant age-related increase in caspase-3 activity; thus the mean value in cortical tissue prepared from aged rats fed on the control diet was significantly increased compared with the value in tissue prepared from young rats (p < 0.05, ANOVA), but EPA suppressed this change. In an effort to establish whether the change in caspase-3 activation was coupled with the increases in IL-1β concentration and p38 activation, a series of in vitro experiments were undertaken that revealed that IL-1β significantly enhanced caspase-3 activity in cortical tissue (p < 0.05, ANOVA; Fig. 3b); this effect was blocked by IL-1ra, suggesting that the action of IL-1β was dependent on receptor interaction, and by SB203580, indicating that the action of IL-1β also required activation of p38. Cells prepared from cortex of young and aged rats fed on both diets were stained for phosphorylated p38 and caspase-3, and staining was assessed using confocal microscopy. We found no cell in preparations obtained from young rats in which there was evidence of colocalization of activated p38 and caspase-3. In contrast, several cells prepared from aged rats fed on the control diet stained positively for both; an example is shown in Fig. 3d. However, while some cells prepared from aged EPA-treated rats stained positively for p38, there was little staining for caspase-3, and we found no evidence of colocalization. These findings support the idea that caspase-3 activation is closely coupled with p38 activation. In addition to the stimulatory effect of IL-1β on caspase-3 activity, we observed that IL-1β significantly increased caspase-3 mRNA in cultured cortical cells (Fig. 3c). In an effort to consolidate these findings, which suggested that there was significant evidence of cell death in the aged cortex, we investigated cleavage of PARP by assessing expression of the 116-kDa form of the enzyme. Fig. 4 indicates, in a sample immunoblot and by analysis of the mean data obtained from densitometric analysis, that there was a significant decrease in expression of 116-kDa PARP in cortical tissue prepared from aged rats fed on the control diet compared with young rats (p < 0.05, ANOVA) but that this effect was reversed in tissue prepared from aged rats treated with EPA. These data were paralleled by changes in TUNEL staining; thus a significantly greater number of cells stained positively for TUNEL in preparations obtained from aged rats fed on the control diet compared with young rats fed on either diet (p < 0.05, ANOVA; Fig.5a). The number of TUNEL-positive cells in preparations obtained from aged rats fed on the EPA-enriched diet was similar to that in tissue prepared from young rats. Fig. 5b shows that exposure of cultured cortical neurons to IL-1β for 72 h also significantly increased the number of TUNEL-positive cells (p < 0.05, Student'st test for independent means).Figure 5The increase in TUNEL staining with age is blocked by EPA and mimicked by IL-1β.a, the number of cells that stained positively for TUNEL was significantly enhanced in cortex of aged rats fed on the control diet (e.g. left-hand picture) compared with that in young rats fed on either diet (*, p < 0.05, ANOVA); dietary manipulation with EPA reversed this effect. b, incubation of cultured cortical neurons in the presence of IL-1β significantly increased the number of TUNEL-positive cells (*,p < 0.05, Student's t test for independent means; n = 5). Con, control; +ve, positive.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In an effort to explore the synaptic changes that might occur as a consequence of IL-1β-induced cell death, we turned to analysis of changes in hippocampus and first assessed age- and diet-related changes in IL-1β concentration and p38 activation. Fig.6a shows that, in parallel with the age-related findings in cortical tissue, IL-1β concentration was significantly increased in hippocampal tissue prepared from aged rats fed on the control diet compared with young rats fed on either diet (p < 0.05, ANOVA). There was no evidence of a similar age-related increase in aged rats fed on the EPA-enriched diet. Analysis of p38 activation revealed a similar pattern; thus there was a significant age-related increase in p38 activity (p < 0.05, ANOVA, aged rats fed on the control diet versus young rats; Fig. 6b), which was not observed in tissue prepared from aged rats fed on the EPA-enriched diet. A likely causal relationship between IL-1β concentration and p38 activation is suggested by the finding that IL-1β significantly increased p38 activity in hippocampus in vitro (Fig. 6c). In a separate series of experiments, in which no dietary manipulation was made, cryostat sections of tissue were prepared from young and aged rats. We observed that there was a marked increase in p38 staining in hippocampus of aged compared with young rats; sample sections are demonstrated in Fig. 6d. Analysis of LTP in dentate gyrus was undertaken in the same rats in which biochemical ana
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