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• W2012632172 abstract "Lipopolysaccharide, a component of the cell wall of Gram-negative bacteria, may be responsible for at least some of the pathophysiological sequelae of bacterial infections, probably by inducing an increase in interleukin-1β (IL-1β) concentration. We report that intraperitoneal injection of lipopolysaccharide increased hippocampal caspase-1 activity and IL-1β concentration; these changes were associated with increased activity of the stress-activated kinase c-Jun NH2-terminal kinase, decreased glutamate release, and impaired long term potentiation. The degenerative changes in hippocampus and entorhinal cortical neurones were consistent with apoptosis because translocation of cytochromec and poly(ADP-ribose) polymerase cleavage were increased. Inhibition of caspase-1 blocked these changes, suggesting that IL-1β mediated the lipopolysaccharide-induced changes. Lipopolysaccharide, a component of the cell wall of Gram-negative bacteria, may be responsible for at least some of the pathophysiological sequelae of bacterial infections, probably by inducing an increase in interleukin-1β (IL-1β) concentration. We report that intraperitoneal injection of lipopolysaccharide increased hippocampal caspase-1 activity and IL-1β concentration; these changes were associated with increased activity of the stress-activated kinase c-Jun NH2-terminal kinase, decreased glutamate release, and impaired long term potentiation. The degenerative changes in hippocampus and entorhinal cortical neurones were consistent with apoptosis because translocation of cytochromec and poly(ADP-ribose) polymerase cleavage were increased. Inhibition of caspase-1 blocked these changes, suggesting that IL-1β mediated the lipopolysaccharide-induced changes. interleukin-1β lipopolysaccharide long term potentiation c-Jun NH2-terminal kinase 2′7′-dichlorofluorescein PBS, phosphate-buffered saline poly(ADP-ribose) polymerase analysis of variance excitatory post-synaptic potential There is increasing awareness of the existence of bidirectional communication between the immune and nervous systems. The proinflammatory cytokine, interleukin-1β (IL-1β),1 is one molecule that may play a pivotal role in integrating neuronal immune responses with those of the endocrine system because it exerts significant effects in all systems, for example in response to stressors such as infection. Gram-negative bacterial infections are associated with multiple pathophysiological changes; it is widely accepted that these changes are stimulated by lipopolysaccharide (LPS), a component of the outer membrane of most Gram-negative bacteria. These changes, which include fever, changes in sleep pattern, and anorexia (1Linthorst A.C.E. Reul J.M.H.M. Ann. N. Y. Acad. Sci. 1998; 840: 139-152Crossref PubMed Scopus (72) Google Scholar), are mimicked by, and therefore thought to be mediated through production of, IL-1β. Thus LPS, injected centrally or peripherally, increases IL-1β concentrations (2Quan N. Sundar S.K. Weiss J.M. J. Neuroimmunol. 1994; 49: 125-134Abstract Full Text PDF PubMed Scopus (207) Google Scholar, 3Van Dam A.M. Poole S. Schultzberg M. Zavala F. Tilders F.J. Ann. N. Y. Acad. Sci. 1998; 840: 128-138Crossref PubMed Scopus (55) Google Scholar) and IL-1β mRNA expression (4Ilyin S.E. Gayle D. Flynn M.C. Plata-Salaman C.R. Brain Res. Bull. 1998; 45: 507-515Crossref PubMed Scopus (63) Google Scholar) in rat brain. Although it appears that in certain circumstances IL-1β may be neuroprotective, the consensus is that prolonged exposure, or exposure of tissue to high concentrations of IL-1β, results in degenerative changes (5Rothwell N.J. J. Physiol. ( Lond. ). 1999; 514: 3-17Crossref PubMed Scopus (218) Google Scholar). Therefore it is significant that increased IL-1 concentrations in different brain areas have been correlated with neurodegenerative disorders such as Down syndrome, Alzheimer's disease (6Griffin 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. A. 1989; 86: 7611-7615Crossref PubMed Scopus (1654) Google Scholar), and Parkinson's disease (7Mogi M. Harada M. Narabayashi H. Inagaki H. Minami M. Nagatsu T. Neurosci Lett. 1996; 211: 13-16Crossref PubMed Scopus (438) Google Scholar), whereas in experimental models, IL-1β is considered to be responsible for the cell damage associated with ischemia (8Ianotti F. Kida S. Weller R. Buhagier G. Hillhouse E. J. Cereb. Blood Flow Metab. 1993; 13 (suppl.): 125Google Scholar) and excitotoxicity (9Panegyres P.K. Hughes J. J. Neurol. Sci. 1998; 154: 123-132Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and is increased after experimental traumatic lesions (10Taupin V. Toulmond S. Serrano A. Benavides J. Zavala F. J. Neuroimmunol. 1993; 42: 177-185Abstract Full Text PDF PubMed Scopus (462) Google Scholar). A striking example of a neuronal deficit induced by IL-1β is the impairment in long term potentiation (LTP) in the hippocampus in vitro (11Bellinger F.P. Madamba S. Siggins G.R. Brain Res. 1993; 628: 227-234Crossref PubMed Scopus (320) Google Scholar, 12Katsuki H. Nakai S. Hirai Y. Akaji K. Kiso Y. Satoh M. Eur. J. Pharmacol. 1990; 181: 323-326Crossref PubMed Scopus (290) Google Scholar, 13Cunningham 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 (350) Google Scholar) and in vivo (14Murray C. Lynch M.A. J. Neurosci. 1998; 18: 2974-2981Crossref PubMed Google Scholar, 15Murray C. Lynch M.A. J. Biol. Chem. 1998; 273: 12161-12168Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 16O'Donnell E. Vereker E. Lynch M.A. Eur. J. Neurosci. 2000; 12: 345-352Crossref PubMed Scopus (115) Google Scholar). IL-1β is produced by glia (17Giulian D. Baker T.J. Shih L.-C.N. Lachman L.B. J. Exp. Med. 1986; 164: 594-604Crossref PubMed Scopus (752) Google Scholar, 18Yao J. Keri J. Taffs R.E. Colton C.A. Brain Res. 1992; 591: 88-93Crossref PubMed Scopus (112) Google Scholar) and neurones (19Farrar W.L. Hill J.M. Harel-Bellan A. Vinocour M. Immunol. Rev. 1987; 100: 361-378Crossref PubMed Scopus (158) Google Scholar, 20Lechan R.M. Toni R. Clark B.D. Cannon J.G. Shaw A.R. Dinarello C.A. Reichlin S. Brain Res. 1990; 514: 135-140Crossref PubMed Scopus (334) Google Scholar) in response to tissue stress. It is cleaved from the inactive percursor, pro-IL-1β, by the action of caspase-1, a member of a large family of cysteine proteases that have been implicated in apoptotic cell death (21Martinou J.-C. Sadoul R. Curr. Opin. Neurobiol. 1996; 6: 609-614Crossref PubMed Scopus (31) Google Scholar, 22Ouyang Y.B. Tan Y. Comb M. Liu C.L. Martone M.E. Siesjo B.K. Ho B.R. J. Cereb. Blood Flow Metab. 1999; 19: 1126-1135Crossref PubMed Scopus (231) Google Scholar, 23Faherty C.J. Xanthoudakis S. Smeyne R.J. Brain Res. Mol. Brain Res. 1999; 18: 159-163Crossref Scopus (100) Google Scholar, 24Becker A.J. Gillardon F. Blumke I. Langendorfer D. Beck H. Westler O.D. Brain Res. Mol. Brain Res. 1999; 67: 172-176Crossref PubMed Scopus (74) Google Scholar, 25Chen J. Nagayama T. Jin K. Stetler R.A. Zhu R.L. Graham S.H. Simon R.P. J. Neurosci. 1999; 18: 4914-4928Crossref Google Scholar). It might be predicted therefore that any trigger such as LPS, which induces an increase in IL-1β, will do so by increasing activity of caspase-1. Our objective was to investigate the cellular consequences of an increase in IL-1β concentration in hippocampus in an effort to establish the mechanism by which IL-1β inhibits LTP in dentate gyrus. Intraperitoneal injection of LPS stimulated caspase-1 activity and induced an increase in IL-1β concentration, and these changes were paralleled by an increase in activity of the stress-activated protein kinase c-Jun NH2-terminal kinase (JNK), a decrease in glutamate release, and inhibition of LTP in perforant path granule cell synapses. These changes, and the degenerative changes in neurones of the hippocampus and entorhinal cortex, were reversed by caspase-1 inhibition. Six groups of six male Wistar rats (250–350 g), obtained from the BioResources Unit, Trinity College Dublin, were anesthetized by intraperitoneal injection of urethane (1.5 g/kg). All rats groups received 1 ml of saline or 1 ml of LPS (200 μg/kg) intraperitoneally; four groups were pretreated either with an intracerebroventricular injection of 5 μl of saline or 5 μl of the caspase-1 inhibitor (10 pmol of Ac-YVAD-CMK, 2.5 mm posterior to Bregma, 0.2 mm lateral to midline, 3.5-mm depth) prior to the intraperitoneal injection and monitored for 3 h. A bipolar stimulating electrode and a unipolar recording electrode were placed in the perforant path (4.4 mm lateral to Lambda) and in the dorsal cell body region of the dentate gyrus (2.5 mm lateral and 3.9 mm posterior to Bregma), respectively, and 0.033-Hz test shocks were given for 10 min before, and 40 min after, tetanic stimulation (three trains of stimuli delivered at 30-s intervals, 250 Hz for 200 ms (26McGahon B. Lynch M.A. Neuroscience. 1996; 72: 847-855Crossref PubMed Scopus (52) Google Scholar)). Rats were killed by cervical dislocation; cross-chopped slices (350 × 350 μm) were prepared from ipsilateral and contralateral dentate gyri, entorhinal cortex, and hippocampus and used to prepare dissociated cells (see below) or frozen separately in Krebs solution containing 10% dimethyl sulfoxide (27Haan E.A. Bowen D.M. J. Neurochem. 1981; 37: 243-246Crossref PubMed Scopus (73) Google Scholar) and stored at −80 °C. For analysis, slices were thawed rapidly and rinsed in fresh oxygenated Krebs solution before preparation of homogenate or the crude synaptosomal pellet P2 (26McGahon B. Lynch M.A. Neuroscience. 1996; 72: 847-855Crossref PubMed Scopus (52) Google Scholar). Formation of reactive oxygen species was assessed by measuring 2′7′-dichlorofluorescein (DCF), the oxidized, fluorescent product of 2′7′-dichlorofluorescein diacetate (DCFH-DA (28Lebel C.P. Bondy S.C. Neurochem. Int. 1990; 17: 435-440Crossref PubMed Scopus (291) Google Scholar)). Synaptosomes prepared from hippocampal slices were incubated at 37 °C for 15 min in the presence of 10 μl of 5 μm DCFH-DA (from a stock of 500 μm) in methanol and centrifuged at 13,000 × g for 8 min at 4 °C to yield pellets that were resuspended in 2 ml of ice-cold 40 mm Tris buffer, pH 7.4, and monitored for fluorescence at 37 °C (excitation, 488 nm; emission, 525 nm). Cleavage of the caspase-1 substrate (YVAD peptide, Alexis Corporation) to its fluorescent product was used as a measure of caspase-1 activity. Slices of tissue were washed, homogenized in 400 μl of lysis buffer (25 mmHEPES, 5 mm MgCl2, 5 mmdithiothreitol, 5 mm EDTA, 2 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin, pH 7.4), subjected to four freeze-thaw cycles, and centrifuged at 15,000 rpm for 20 min at 4 °C. 90-μl samples of supernatant were added to 10 μl of 500 μm YVAD peptide and incubated at 37 °C for 60 min. 900 μl of incubation buffer (100 mm HEPES containing 10 mm dithiothreitol, pH 7.4) was added, and fluorescence was assessed (excitation, 400 nm; emission, 505 nm). IL-1β concentration in homogenate prepared from hippocampus of entorhinal cortex was analyzed by enzyme-linked immunosorbent assay (14; Genzyme Diagnostics). Antibody-coated (2.0 μg/ml final concentration, diluted in 0.1m sodium carbonate buffer, pH 9.5; monoclonal hamster anti-mouse IL-1β antibody) 96-well plates were incubated overnight at 4 °C, washed several times with phosphate-buffered saline (PBS) containing 0.05% Tween 20, blocked for 2 h at 37 °C with 250 μl of blocking buffer (0.1 m PBS, pH 7.3, with 4% bovine serum albumin), and incubated with 50-μl IL-1β standards (0–1,000 pg/ml) for 1 h at 37 °C. Samples were incubated with 100 μl of secondary antibody (final concentration 0.8 μg/ml in PBS containing 0.05% Tween 20 and 1% bovine serum albumin; biotinylated polyclonal rabbit anti-mouse IL-1β antibody) for 1 h at 37 °C, washed, and incubated in 100 μl of detection agent (horseradish peroxidase-conjugated streptavidin; 1:1,000 dilution in PBS containing 0.05% Tween 20 and 1% bovine serum albumin) for 15 min at 37 °C. 100 μl of tetramethylbenzidine (Sigma) was added, incubated at room temperature for 10 min, and absorbance read at 450 nm within 30 min. JNK phosphorylation was analyzed in samples prepared from hippocampal tissue; cytochromec translocation and PARP cleavage were analyzed in samples prepared from entorhinal cortex. In the case of JNK and PARP, tissue homogenates were diluted to equalize for protein concentration (29Bradford M.M. Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar), and 10-μl aliquots (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% bromphenol blue, w/v), boiled for 5 min, and loaded onto gels (10% SDS for PARP and 12% for JNK). In the case of cytochrome c, the cytosolic fraction was prepared by homogenizing slices of entorhinal cortex in lysis buffer (composition in mm: 20 HEPES, pH 7.4, 10 KCl, 1.5 MgCl2, 1 EGTA, 1 EDTA, 1 dithiothreitol, 0.1 phenylmethylsulfonyl fluoride, 5 μg/ml pepstatin A, 2 μg/ml leupeptin, 2 μg/ml aprotonin), 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 mmTris-HCl, pH 6.8, 10% glycerol v/v, 4% SDS w/v, 5% β-mercaptoethanol v/v, 0.002% bromphenol blue w/v) 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 a 30-mA constant current for 25–30 min, transferred onto nitrocellulose strips (225 mA for 75 min), and immunoblotted with the appropriate antibody. To assess JNK activity, proteins were immunoblotted with an antibody that specifically targets phosphorylated JNK (Santa Cruz Biotechnology, Inc.; 1:2,000 in PBS and 0.1% Tween 20 containing 2% non-fat dried milk) for 2 h at room temperature. Immunoreactive bands were detected using peroxidase-conjugated anti-mouse IgG (Sigma) and enhanced chemiluminescence (Amersham Pharmacia Biotech). To assess cleavage of PARP, we immunoblotted with an antibody (1:2,000) raised against the epitope corresponding to amino acids 764–1014 of PARP of human origin (Santa Cruz Biotechnology Inc.), and immunoreactive bands were detected using peroxidase-conjugated anti-rabbit IgG (Sigma) and enhanced chemiluminescence. To assess cytochrome c, a rabbit polyclonal antibody raised against recombinant protein corresponding to amino acids 1–104 of cytochrome c (Santa Cruz Biotechnology Inc.) was used. Immunoreactive bands were detected using peroxidase-conjugated anti-rabbit antibody (Sigma) and enhanced chemiluminescence. Synaptosomal tissue prepared from untetanized and tetanized dentate gyrus was resuspended in ice-cold Krebs solution containing 2 mm CaCl2, aliquotted onto 0.45-μm Millipore filters, and rinsed under vacuum. Tissue was incubated in 250 μl of oxygenated Krebs solution ± 40 mm KCl at 37 °C for 3 min, and the filtrate was collected and stored. To analyze glutamate concentration, triplicate 50-μl samples or 50-μl glutamate standards (50 nm to 10 μm in 100 mm Na2HPO4buffer, pH 8.0) were added to 320-μl glutaraldehyde (0.5% in 100 mm NaH2PO4 buffer, pH 4.5)-coated 96-well plates and incubated for 60 min at 37 °C (30Ordronneau P. Abdullah L. Petruse P. J. Immunol. Methods. 1991; 142: 169-176Crossref PubMed Scopus (41) Google Scholar). 250 μl of ethanolamine (0.1 m in 100 mmNa2HPO4 buffer) and 200 μl of donkey serum (3% in PBS-T) were used to bind unreacted aldehydes and to block nonspecific binding, respectively. Samples were incubated overnight at 4 °C in the presence of 100 μl of anti-glutamate antibody (raised in rabbit, 1:5,000 in PBS-T, Sigma), washed with PBS-T, and then incubated for 60 min at room temperature with 100 μl of anti-rabbit horseradish peroxidase-linked secondary antibody (1:10,000 in PBS-T, Amersham Pharmacia Biotech). 100 μl of 3,3′,5,5′-tetramethylbenzidine liquid substrate was added, incubation continued for exactly 60 min, 30 μl of 4 m H2SO4 was added to stop the reaction, and optical densities were determined at 450 nm. 350-μm slices prepared from entorhinal cortex and hippocampus were equilibrated in oxygenated Krebs solution for 30 min at 30 °C and then incubated in Krebs solution containing 1 mg/ml protease X, 1 mg/ml protease XIV, and 1,600 Kunitz Dnase for 30 min at 30 °C. Washed slices were resuspended in 1 ml of prewarmed Dulbecco's modified essential medium containing 1,600 Kunitz Dnase, triturated with a glass Pasteur pipette, and passed through a nylon mesh filter to remove tissue clumps. 30-μl aliquots were plated out on poly-l-lysine-coated 11-mm round glass coverslips, placed in a 5% CO2 incubator at 37 °C for 1 h, and fixed in 4% paraformaldehyde (30 min at room temperature). Coverslips were stored at 4 °C in PBS until use (31Mody I. Slater M.W. MacDonald J.F. Neurosci. Lett. 1989; 96: 70-75Crossref PubMed Scopus (34) Google Scholar). Cells were stained using the Rapi-diff II staining procedure (DiaCheM International Ltd., Lancastershire, U. K,) and viewed under × 100 magnification. Cells displaying degenerative features (e.g. shrinkage and membrane blebbing) were counted and expressed as a percentage of the total number of cells examined (80–100/coverslip in the case of entorhinal cortex and 100–200 in the case of the hippocampus). A one-way analysis of variance (ANOVA) was performed to determine whether there were significant differences between conditions. When this analysis indicated significance (at the 0.05 level), post hoc Student Newmann-Keuls test analysis was used to determine which conditions were significantly different from each other. Student's t test was used to establish statistical significance in some cases; for example, when analysis was performed on tissue prepared from untetanized and tetanized tissue obtained from the same rat. Tetanic stimulation delivered to the perforant path 3 h after intraperitoneal injection of LPS resulted in an increase in the mean slope of the population epsp; the mean percentage increase in the 2 min immediately following tetanic stimulation (± S.E., compared with the 5 min immediately before tetanic stimulation) was 133.58 (± 3.48), but this was not maintained so that the mean percentage increase in population epsp slope in the last 5 min of the experiment was 100.81 ± 2.26. The corresponding values in the saline-treated control rats were 164.83 ± 4.23 and 119.1 ± 2.17, respectively (Fig.1; n = 6 in both groups). The stimulus strength required to induce a spike was 7.87V (± 0.98) in LPS-treated rats compared with 4.02V ± 1.2 in saline-injected rats. The LPS-induced attenuated LTP was associated with a significant increase in reactive oxygen species production, caspase-1 activity, IL-1β concentration, and JNK activity (*p < 0.05, **p < 0.01, Student's t test for independent means, Fig. 2, a–d;n = 6 in all cases) in hippocampus. The stimulatory effect of LPS on JNK activity was mimicked by the addition of IL-1β to hippocampal tissue in vitro (p < 0.01, Student's t test for paired means, Fig. 2 e). Fig. 2 f indicates that endogenous glutamate release was increased significantly by the addition of 40 mm KCl to synaptosomes prepared from untetanized dentate gyrus of saline-pretreated rats (*p < 0.05, Student'st test for paired means), but this effect was enhanced in synaptosomes prepared from tetanized dentate gyrus (**p< 0.01, Student's t test for paired means). In contrast, KCl failed to stimulate glutamate release in synaptosomes prepared from untetanized dentate gyrus of LPS-pretreated rats, although release was increase in tetanized tissue (*p < 0.05, Student'st test for paired means) albeit to an attenuated degree. These data suggested that the LPS-induced effect on LTP may be a consequence of its ability to increase activity of caspase-1 and thence IL-1β concentration and to determine whether this was the case, rats were injected intracerebroventricularly with 5 μl of a caspase-1 inhibitor peptide (Ac-YVAD-CMK) or with 5 μl of saline prior to LPS or saline treatment. Fig. 3 a indicates that although LTP was inhibited by LPS, this effect was blocked by the caspase-1 inhibitor. Thus the mean percentage change in population epsp slope (± S.E.) in the 2 min immediately after tetanic stimulation was 177.77 ± 15.34 in the control group (treated with saline intracerebroventricularly and intraperitoneally) compared with 118.92 ± 3.35 in the group treated with saline intracerebroventricularly and LPS intraperitoneally. In the last 5 min of the experiment the values were 123.86 ± 2.14 and 96.21 ± 1.14, respectively. Injection of the caspase-1 inhibitor partially reversed the inhibitory effect of LPS on the early changes induced by the tetani and completely blocked the LPS-induced inhibition of the later phase of LTP; the mean percentage changes were 147.32 ± 9.23 and 123.54 ± 6.33 in the first 2 min after tetanic stimulation and in the last 5 min of the experiment, respectively. However, LTP was similar in the control rats and the group of rats injected the caspase-1 inhibitor intracerebroventricularly and saline intraperitoneally; in the latter group, the mean percentage changes in population epsp slope were 174.57 ± 16.24 and 142.07 ± 7.42 in the 2 min after tetanic stimulation and the last 5 min of the experiment, respectively (Fig. 3 a, n = 6 in all groups). Fig. 3 b shows that intraperitoneal injection of LPS (in rats treated with saline intracerebroventricularly) significantly increased IL-1β concentration in hippocampus (* p < 0.05, Student's t test for independent means, n = 6) and that this effect was inhibited by pretreatment with the caspase-1 inhibitor. Similarly, JNK activity was enhanced significantly in hippocampal tissue prepared from LPS-treated rats (p< 0.05, Student's t test for independent means), but this effect was also inhibited by the caspase-1 inhibitor (Fig.3 c). Acutely dissociated cells were prepared from hippocampal tissue obtained from rats in each of the four treatment groups. LPS treatment significantly increased the number of degenerating cells, with evidence of an increased number of cells displaying degenerative features such as shrinkage and blebbing of the plasma membrane (Fig. 4 b). This contrasts with cells prepared from saline-treated rats (panel a) and rats treated only with caspase-1 inhibitor (panel c). Treatment with the caspase-1 inhibitor (panel d) partially reversed the effects of LPS with fewer cells displaying degenerative changes. Fig. 4 e shows that the percentage of cells which showed degenerative changes was enhanced significantly in the LPS-treated group compared with any of the other groups (p < 0.05, Student's t test for independent means) and indicates that the caspase-1 inhibitor reversed the degenerative effect of LPS. In an effort to account for the compromise in transmitter release observed in dentate gyrus synaptosomes prepared from LPS-treated rats, we analyzed caspase-1 activity and IL-1β concentrations in tissue prepared from entorhinal cortex and found that both measures were increased significantly after LPS treatment (*p < 0.05, Student's t test for independent means), but these effects were both attenuated by the caspase-1 inhibitor (Fig.5, a and b,n = 6). In parallel with the observations in hippocampus, we observed that there was an LPS-induced increase in the number of degenerating cells changes (** p < 0.01, Student's t test for independent means, Fig.6 a), with evidence of cell shrinkage and membrane blebbing. Pretreatment with the caspase-1 inhibitor blocked these LPS-associated changes (Fig. 6, aand b). Consistent with the evidence of cell degeneration in hippocampus, we observed that cytochrome c translocation was increased markedly in tissue prepared from entorhinal cortex of LPS-treated rats (p < 0.01, Student's ttest for independent means), whereas there was a decrease in expression of the 116-kDa fragment of (PARP, * p < 0.05, Student's t test for independent means); both of these LPS-associated changes were attenuated by pretreatment with the caspase-1 inhibitor (Fig. 6, c and d;n = 6).Figure 6The LPS-induced changes in cytochromec, PARP cleavage, and degenerative changes in entorhinal cortex are blocked by the caspase-1 inhibitor. LPS treatment (see panel b, ii) increased the percentage of degenerating cells, exhibiting evidence of degeneration such as shrinkage and blebbing of the plasma membrane (seearrows) compared with saline-treated rats (panel b, i) or rats treated only with caspase-1 inhibitor (panel b, iii); these effects were blocked in rats pretreated with the caspase-1 inhibitor (panel b,iv). The mean data were obtained by counting 80–100 cells on each coverslip (*p < 0.05, Student's ttest for independent means, n = 6). Expression of the 116-kDa subunit of PARP (panel c) was decreased significantly and cytochrome c translocation (panel d) was increased significantly in tissue prepared from entorhinal cortex of LPS-treated rats (*p < 0.05, Student'st test for independent means, n = 6). These effects were blocked in rats pretreated with the caspase-1 inhibitor. Sample immunoblots demonstrate these effects of LPS (lanes 2and 4) compared with control (lanes 1 and3) in saline-pretreated (lanes 1 and2) and Ac-YVAD-CMK-pretreated (lanes 3 and4) rats.View Large Image Figure ViewerDownload (PPT) We set out to investigate the effect of an intraperitoneal injection of LPS on synaptic function in hippocampus because LPS is considered to contribute significantly to the neuropathological effects associated with Gram-negative bacterial infections probably by increasing IL-1β concentration in brain. The evidence presented indicates that the LPS-induced increase in IL-1β concentration, consequent on increased caspase-1 activity, leads to activation of JNK which may underlie the observed decrease in transmitter release in dentate gyrus, degenerative changes in hippocampus and entorhinal cortex, and inhibition of LTP. Intraperitoneal injection of LPS inhibited LTP in perforant path granule cell synapses; to our knowledge this effect of LPS has not been shown previously. The current data present at least two possible mechanisms that might underlie the effects. First, we observed that LPS induced an increase in reactive oxygen species production in hippocampus, and the attenuated LTP may arise, directly or indirectly, from this. Such an effect of oxygen radicals has been reported in CA1in vitro (32Pellmar T.C. Hollinden G.E. Sarvey J.M. Neuroscience. 1991; 44: 353-359Crossref PubMed Scopus (81) Google Scholar), and we have recently observed that LTP in dentate gyrus in vivo was inhibited by hydrogen peroxide. 2A. Lynch, P. M., Queenan, and M. A. Lynch, unpublished observation. A second possibility is that the impairment in LTP is a consequence of the LPS-induced increase in IL-1β concentration in hippocampus. We have observed that intracerebroventricular injection of IL-1β inhibits LTP in perforant path-granule cell synapses in vivo (14Murray C. Lynch M.A. J. Neurosci. 1998; 18: 2974-2981Crossref PubMed Google Scholar, 15Murray C. Lynch M.A. J. Biol. Chem. 1998; 273: 12161-12168Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 33Lynch M.A. Prog. Neurobiol. 1998; 56: 1-19Crossref PubMed Scopus (145) Google Scholar) and that LTP was also compromised in aged and stressed rats, in which hippocampal IL-1β concentration is increased (14Murray C. Lynch M.A. J. Neurosci. 1998; 18: 2974-2981Crossref PubMed Google Scholar). The inhibitory effect of IL-1β on LTP in vitro has also been documented; thus IL-1β-induced attenuation of LTP in CA1 (11Bellinger F.P. Madamba S. Siggins G.R. Brain Res. 1993; 628: 227-234Crossref PubMed Scopus (320) Google Scholar), CA3 (12Katsuki H. Nakai S. Hirai Y. Akaji K. Kiso Y. Satoh M. Eur. J. Pharmacol. 1990; 181: 323-326Crossref PubMed Scopus (290) Google Scholar) and dentate gyrus (13Cunningham 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 (350) Google Scholar) has been reported. Peripheral injection of LPS induced an increase in IL-1β concentration in hippocampus, which supports earlier reports of a similar change in hippocampus and cortex (3Van Dam A.M. Poole S. Schultzberg M. Zavala F. Tilders F.J. Ann. N. Y. Acad. Sci. 1998; 840: 128-138Crossref PubMed Scopus (55) Google Scholar, 34Nguyen K.T. Deak T. Owens S.M. Kohno T. Fleshner M. Watkins L.R. Maier S.F. J. Neurosci. 1998; 18: 2239-2246Crossref PubMed Google Scholar), cerebellum (4Ilyin S.E. Gayle D. Flynn M.C. Plata-Salaman C.R. Brain Res. Bull. 1998; 45: 507-515Crossref PubMed Scopus (63) Google Scholar), and in whole brain (2Quan N. Sundar S.K. Weiss J.M. J. Neuroimmunol. 1994; 49: 125-134Abstract Full Text PDF PubMed Scopus (207) Google Scholar). These data are backed up by several observations of LPS-induced increases in IL-1β concentrations or IL-1β mRNA in cultured glial cells (4Ilyin S.E. Gayle D. Flynn M.C. Plata-Salaman C.R. Brain Res. Bull. 1998; 45: 507-515Crossref PubMed Scopus (63) Google Scholar, 35Kong L.-Y. Lai C. Wilson B.C. Simpson J.N. Hong J.-S. Neurochem. Int. 1997; 30: 491-497Crossref PubMed Scopus (44) Google Scholar), which, together with other data indicating that IL-1β is synthesized in glia (17Giulian D. Baker T.J. Shih L.-C.N. Lachman L.B. J. Exp. Med. 1986; 164: 594-604Crossref PubMed Scopus (752) Google Scholar, 18Yao J. Keri J. Taffs R.E. Colton C.A. Brain Res. 1992; 591: 88-93Crossref PubMed Scopus (112) Google Scholar) and neurons (19Farrar W.L. Hill J.M. Harel-Bellan A. Vinocour M. Immunol. Rev. 1987; 100: 361-378Crossref PubMed Scopus (158) Google Scholar, 20Lechan R.M. To" @default.
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• W2012632172 title "Lipopolysaccharide Inhibits Long Term Potentiation in the Rat Dentate Gyrus by Activating Caspase-1" @default.
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