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Lipopolysaccharide pre-treatment induces resistance against subsequent focal cerebral ischemic damage in spontaneously hypertensive rats
Brain Research 748 Ž1997. 267–270
Short communication
Lipopolysaccharide pre-treatment induces resistance against subsequent focal cerebral ischemic damage in spontaneously hypertensive rats Kaoru Tasaki a , Christl A. Ruetzler a , Toshiho Ohtsuki a , David Martin b , Hiroshi Nawashiro a , John M. Hallenbeck a, ) a
Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA b Department of Pharmacology, Amgen Boulder, Inc. Boulder, CO, USA Accepted 26 November 1996
Abstract Ischemic tolerance was induced in spontaneously hypertensive rats ŽSHR. by injection of a single dose of lipopolysaccharide ŽLPS. Ž0.9 mgrkg, i.v.. 1–7 days prior to permanent middle cerebral artery occlusion ŽMCAO.. Infarct volume, evaluated 24 h after MCAO, was significantly reduced by LPS administration 2, 3 or 4 days prior to MCAO Ž22.8, 25.9 and 20.5%, respectively.. The beneficial effect of LPS pre-treatment was completely nullified by concurrent administration of TNFbp. On this basis, the tolerance to ischemia induced by LPS is likely to be mediated by TNF-a . Keywords: Tolerance; Lipopolysaccharide; Tumor necrosis factor a ; Endotoxin; Cerebral ischemia; Rat
Tolerance to experimental brain ischemia can be induced by a variety of stimuli that elicit a stress response w15,17x. The cytokines, IL-1 and TNF-a , activate intracellular signaling pathways that mediate the stress response w16x and could, therefore, function in ligand-receptor interactions that induce the tolerant state. To explore the possibility that cytokines have a role in the development of tolerance, LPS which elicits the release of IL-1 and TNF-a w14x was administered to spontaneously hypertensive rats ŽSHR. prior to MCAO. IL-1 receptor antagonist ŽIL-1ra. or TNF-binding protein ŽTNFbp. were subsequently co-administered with LPS to assess their capacity to counteract the LPS effects on ischemic brain damage. All animal studies were approved by the National Institute of Neurological Disorders and Stroke Animal Care and Use Committee. In 98 adult male SHR Ž300–350 g., the left middle cerebral and left common carotid arteries were occluded by a modified tandem occlusion technique w2x under 1–1.5% halothane in 30% O 2 and 70% N2 O. Briefly, the left common carotid artery was ligated and the left middle cerebral artery was exposed and electrocoagu-
) Corresponding author. Stroke Branch, NINDS, NIH, Building 36, Room 4A03, 36 Convent Drive MSC 4128, Bethesda, MD 20892-4128, USA. Fax: q1 Ž301. 402-2769.
lated just distal to the inferior cerebral vein; both vessels were transected. Body temperature was maintained at 37.58C with a heating pad during and 2 h after surgery. The left femoral artery was cannulated for blood sampling and monitoring of blood pressure. 24 h after MCAO, animals were deeply anesthetized with pentobarbital and decapitated. Brains were removed and sequential 2 mm coronal brain sections were incubated at 378C in 2% 2,3,5-triphenyltetrazolium chloride ŽTTC. w9x. The total area of each coronal brain section and the lesion area were image-analyzed and lesion volumes were calculated by integration of the section areas. All analyses were performed in a blinded fashion. The time course for the effects on ischemic brain damage of LPS administered as a single dose was investigated. LPS from Escherichia coli 0111:B4 phenol extract ŽSigma, St. Louis, MO. was dissolved in sterile, pyrogenfree 0.9% Žwrv. NaCl and injected i.v. Ž0.9 mgrkg. 1, 2, 3, 4 or 7 days prior to MCAO. This optimal dose was selected from a series of preliminary dose-finding studies. Control animals were injected with the same volume of sterile, pyrogen-free 0.9% saline at the same time points. In separate experiments, the effect of systemic injection of LPS combined with either IL-1ra or TNFbp on ischemic brain damage was investigated. IL-1ra, a gift from Amgen ŽBoulder, CO., was dissolved in 10 mM sodium citrate
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 3 8 3 - 2
K. Tasaki et al.r Brain Research 748 (1997) 267–270
268
buffer, pH 6.8, with 150 mM NaCl, 0.5 mM EDTA and 0.1% Tween-80 and injected s.c. 100 mgrkg immediately before and 4 h after LPS injection Ž n s 4.. In another group of animals, human recombinant type I soluble TNF receptor dimer conjugated to polyethylene glycol to enhance its activity, TNFbp, a gift from Amgen ŽBoulder, CO., was dissolved in 10 mM sodium phosphate, 20 mM sodium chloride, pH 6.5 and injected i.v. 2 mgrkg just before LPS injection Ž n s 5.. Control animals for both groups were injected with LPS in combination with an appropriate volume of each vehicle Ž n s 4 and n s 5, respectively.. MCAO was performed 3 days after the LPS q IL-1ra, LPS q TNFbp or LPS q vehicle injection and the infarct volume was measured 24 h post-MCAO by TTC staining. Four subsequent animals received only TNFbp Ž2 mgrkg i.v.. 72 h before MCAO. Serum TNF-a levels were measured by L929 cell lysis bioassay w8x 2 h after LPS q TNFbp or LPS q vehicle injection. Data are presented as mean " S.D. LPS vs. Saline comparisons at each time point and comparison of LPS q IL-1ra vehicle with LPS q IL-1ra were analyzed by independent groups Student’s t-test. The Welch approximation was used when group variances were heterogenous. The TNFbp study was analyzed by one-way ANOVA and pair-wise group comparisons were made with a Bonferroni correction. Student’s t-test was also used to compare the two serum TNF-a levels. Body temperature was not significantly altered after LPS injection and remained - 398C in all animals. Complete blood cell counts revealed no statistically significant differences between the LPS-injected groups and their respective control groups. There were no significant differences in the physiologic variables in any of the LPS- or vehicle-pre-treated groups at any of the 5 time points ŽTable 1.. Similarly, there were no differences in physiologic variables in the blocking studies with LPS q IL-1ra, LPS q TNFbp or their respective controls.
Fig. 1. Infarct volumes: LPS Ž0.9 mgrkg. or saline i.v. at several times before MCAO. ) P - 0.05; ) ) P - 0.02.
Pre-treatment with LPS at 2, 3 or 4 days prior to MCAO reduced the volume of the ischemic damage by 22.8% Ž P - 0.05., 25.9% Ž P - 0.02. and 20.5% Ž P 0.02., respectively. Infarct volumes expressed in mm3 in the 5 groups that constitute the time-course experiments were: saliner1 day 162 " 22, LPSr1 day 138 " 30, saliner2 days 163 " 35, LPSr2 days 122 " 23, saliner3 days 162 " 37, LPSr3 days 116 " 26, saliner4 days 163 " 12, LPSr4 days 128 " 27, saliner7 days 165 " 11, LPSr7 days 163 " 25 ŽFig. 1.. In the blocking studies, ischemic lesion volume was not significantly different between the LPS q IL-1ra-treated group Ž109 " 48 mm3 . and the control group Ž118 " 42 mm3 .. The infarct volume produced by LPS q TNFbp treatment Ž174 " 24 mm3 . was significantly larger than that of its control group Ž119 " 23 mm3, P - 0.03. ŽFig. 2. and was essentially the same volume as that produced by saline pre-treatment. TNFbp pre-treatment alone Ž131 " 19 mm3 . did not increase in-
Table 1 Physiologic variables at occlusion of the middle cerebral artery Time of LPS administration Žpre-MCAO. 1 day LPS Ž n s 8. Saline Ž n s 8. 2 days LPS Ž n s 8. Saline Ž n s 8. 3 days LPS Ž n s 8. Saline Ž n s 8. 4 days LPS Ž n s 8. Saline Ž n s 8. 7 days LPS Ž n s 8. Saline Ž n s 8.
pO 2 Žmm Hg.
pCO 2 Žmm Hg.
pH
Hematocrit Ž%.
Glucose Žmgr100 ml.
MABP Žmm Hg.
155 " 10 136 " 21
39 " 3 39 " 2
7.38 " 0.01 7.42 " 0.02
41 " 2 42 " 1
123 " 12 130 " 11
130 " 12 142 " 7
128 " 38 120 " 16
44 " 2 41 " 4
7.38 " 0.02 7.40 " 0.01
47 " 3 43 " 3
123 " 12 126 " 16
129 " 12 136 " 12
122 " 19 120 " 10
40 " 4 42 " 4
7.40 " 0.01 7.40 " 0.02
44 " 4 46 " 3
121 " 17 130 " 27
132 " 9 138 " 8
142 " 15 133 " 15
41 " 5 38 " 2
7.40 " 0.02 7.41 " 0.02
39 " 2 40 " 4
146 " 17 144 " 10
129 " 10 141 " 11
136 " 15 132 " 22
40 " 2 38 " 5
7.40 " 0.02 7.40 " 0.02
38 " 2 42 " 3
122 " 13 124 " 11
142 " 7 148 " 9
K. Tasaki et al.r Brain Research 748 (1997) 267–270
269
Fig. 2. Infarct volumes: ŽA. MCAO 3 days after LPS q vehicle or LPS q IL-1ra injections; ŽB. MCAO 3 days after LPS q vehicle, LPS q TNFbp, TNFbp only, or saline only injection. ) P - 0.03.
farct volume compared to saline pre-treatment Ž162 " 37 mm3 .. Thus, administration of TNFbp completely nullified the protective effect of LPS pre-treatment. Serum TNF-a levels were measured in an LPS q vehicle group Ž307, 200 " 253, 599 Urml, n s 4. and TNFbp injection coincident with LPS suppressed the serum TNF-a activity Ž80 " 0 Urml, n s 4, P - 0.05.. The present findings demonstrate that systemic LPS administration 48–96 h prior to MCAO induces resistance to subsequent ischemic neuronal damage in SHR. TNFbp, which was demonstrated to greatly inhibit the bioactivity of TNF-a after LPS administration, nullified this effect, indicating that pre-ischemic elevation of TNF-a activity by the LPS is necessary to induce the state of tolerance to subsequent ischemia. Although the LPS was systemically administered and its effects on the cerebral vasculature and circulating cells may account for much of the induced tolerance, systemic LPS also has central actions and can induce TNF-a production by microglia and astrocytes. This raises the possibility that brain parenchymal cells participate in the development of tolerance in this model w1,7,11x. TNF-a pre-treatment has been observed to reduce myocardial damage during perfusion arrest in a Langendorff preparation w5x and lethal decompression sickness in SHR w4x. This is the first demonstration that pre-ischemic administration of LPS induces tolerance to focal brain ischemia. IL-1ra was administered in a dose that has been demonstrated to be neuroprotective during focal brain ischemia in rats w6x. The half-life of IL-1ra is f 6 h after i.p. injection w12x. The biologic activity of the drug in our experimental paradigm was, therefore, probably no longer than 10–12 h after LPS adminmistration. It is possible that continued administration of IL-1ra for several days to more closely match the duration of action of the TNFbp ŽT1r2 24–36 h. w13x would also have demonstrated some attenuation of LPS tolerance induction and, thus, implicated IL-1 in addition to TNF-a as a mediator of this tolerance.
Since our work suggests that tolerance to brain ischemia proceeds from activation of TNF-a receptors, this should greatly facilitate characterization of the intracellular signaling pathways that regulate the tolerant state. This work implicates the p55 and p75 TNF receptors w18x. Activation of TNF receptors leading to production of sphingomyelinbased second messengers and activation of stress-activated protein kinases ŽSPAKs. are, therefore, a potentially relevant series of intracellular responses w10x. Nuclear translocation of NF-k B may also be involved in the stress-induced tolerance observed in this model w3x. Intracellular signaling pathways that originate from TNF-a receptors merit further attention as candidate mechanisms for the regulation of tolerance to ischemia. Acknowledgements We are grateful to Dr. Anna-Leena Siren ´ for measurement of serum TNF-a and to Karen Pettigrew for expert statistical consultation. References w1x Breder, C.D., Hazuka, C., Ghayur, T., Klug, C., Huginin, M., Yasuda, K., Teng, M. and Saper, C.B., Regional induction of tumor necrosis factor a expression in the mouse brain after systemic lipopolysaccharide administration, Proc. Natl. Acad. Sci. USA, 91 Ž1994. 11393–11397. w2x Brint, S., Jacewicz, M., Kiessling, M., Tanabe, J. and Pulsinelli, W., Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries, J. Cereb. Blood Flow Metab., 8 Ž1988. 474–485. w3x Claudio, E., Segade, F., Wrobel, K., Ramos, S., Bravo, R. and Lazo, P.S., Molecular mechanisms of TNF-a cytotoxicity: activation of NF-k B and nuclear translocation, Exp. Cell Res., 224 Ž1996. 63–71. w4x Dutka, A.J. and Broome, J. Spontaneously hypertensive rats ŽSHR. tolerant to endotoxin do not develop decompression illness, Undersea Hyperbaric Med., 21 ŽSuppl.. Ž1994. 92–93. w5x Eddy, L.J., Goeddel, D.V. and Wong, G.H.W., Tumor necrosis factor-a pretreatment is protective in a rat model of myocardial
270
w6x
w7x
w8x
w9x
w10x
w11x
K. Tasaki et al.r Brain Research 748 (1997) 267–270 ischemia-reperfusion injury, Biochem. Biophys. Res. Commun., 184 Ž1992. 1056–1059. Garcia, J.H., Liu, K.-F. and Relton, J.K., Interleukin-1 receptor antagonist decreases the number of necrotic neurons in rats with middle cerebral artery occlusion, Am. J. Pathol., 147 Ž1995. 1477– 1486. Gutierrez, E.G., Banks, W.A. and Kastin, A.J., Murine tumor necrosis factor alpha is transported from blood to brain in the mouse, J. Neuroimmunol., 47 Ž1993. 169–176. Hogan, M.M. and Vogel, S.N., Production of tumor necrosis factor by rIFN-g-primed C3HrHeJ ŽLPSd. macrophages requires the presence of lipid A-associated proteins, J. Immunol., 141 Ž1988. 4196– 4202. Isayama, K., Pitts, L.H. and Nishimura, M.C., Evaluation of 2,3,5triphenyltetrazolium chloride staining to delineate rat brain infarcts, Stroke, 22 Ž1991. 1394–1398. Kyriakis, J.M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E.A., Ahmad, M.F., Avruch, J. and Woodgett, J.R., The stress-activated protein kinase subfamily of c-jun kinases, Nature, 369 Ž1994. 156–160. Laye, ´ S., Parnet, P., Goujon, E. and Dantzer, R., Peripheral administration of lipopolysaccharide induces the expression of cytokine transcripts in the brain and pituitary of mice, Mol. Brain. Res., 27 Ž1994. 157–162.
w12x Martin, D., Chinookoswong, N. and Millfer, G., The interleukin-1 antagonist ŽrhIL-1ra. protects against cerebral infarction in a rat model of hypoxia-ischemia, Exp. Neurol., 130 Ž1994. 362–367. w13x Martin, D., Near, S.L., Bendele, A. and Russell, D.A., Inhibition of tumor necrosis factor is protective against neurologic dysfunction after active immunization of Lewis rats with myelin basic protein, Exp. Neurol., 131 Ž1995. 221–228. w14x Pugin, J., Ulevitch, R.J. and Tobias, P.S., Tumor necrosis factor-a and interleukin-1 b mediate human endothelial cell activation in blood at low endotoxin concentrations, J. Inflammat., 45 Ž1995. 49–55. w15x Sakaki, T., Yamada, K., Otsuki, H., Yuguchi, T., Kohmura, E. and Hayakawa, T., Brief exposure to hypoxia induces bFGF mRNA and protein and protects rat cortical neurons from prolonged hypoxic stress, Neurosci. Res., 23 Ž1995. 289–296. w16x Schobitz, B., De Kloet, E.R. and Holsboer, F., Gene expression and ¨ function of Interleukin 1, interleukin 6 and tumor necrosis factor in the brain, Prog. Neurobiol., 44 Ž1994. 397–432. w17x Simon, R.P., Niiro, M. and Gwinn, R., Prior ischemic stress protects against experimental stroke, Neurosci. Lett., 163 Ž1993. 135–137. w18x Vilcek, J. and Lee, T.H., Tumor necrosis factor. New insights into the molecular mechanisms of its multiple actions, J. Biol. Chem., 266 Ž1991. 7313–7316.
Short communication
Lipopolysaccharide pre-treatment induces resistance against subsequent focal cerebral ischemic damage in spontaneously hypertensive rats Kaoru Tasaki a , Christl A. Ruetzler a , Toshiho Ohtsuki a , David Martin b , Hiroshi Nawashiro a , John M. Hallenbeck a, ) a
Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA b Department of Pharmacology, Amgen Boulder, Inc. Boulder, CO, USA Accepted 26 November 1996
Abstract Ischemic tolerance was induced in spontaneously hypertensive rats ŽSHR. by injection of a single dose of lipopolysaccharide ŽLPS. Ž0.9 mgrkg, i.v.. 1–7 days prior to permanent middle cerebral artery occlusion ŽMCAO.. Infarct volume, evaluated 24 h after MCAO, was significantly reduced by LPS administration 2, 3 or 4 days prior to MCAO Ž22.8, 25.9 and 20.5%, respectively.. The beneficial effect of LPS pre-treatment was completely nullified by concurrent administration of TNFbp. On this basis, the tolerance to ischemia induced by LPS is likely to be mediated by TNF-a . Keywords: Tolerance; Lipopolysaccharide; Tumor necrosis factor a ; Endotoxin; Cerebral ischemia; Rat
Tolerance to experimental brain ischemia can be induced by a variety of stimuli that elicit a stress response w15,17x. The cytokines, IL-1 and TNF-a , activate intracellular signaling pathways that mediate the stress response w16x and could, therefore, function in ligand-receptor interactions that induce the tolerant state. To explore the possibility that cytokines have a role in the development of tolerance, LPS which elicits the release of IL-1 and TNF-a w14x was administered to spontaneously hypertensive rats ŽSHR. prior to MCAO. IL-1 receptor antagonist ŽIL-1ra. or TNF-binding protein ŽTNFbp. were subsequently co-administered with LPS to assess their capacity to counteract the LPS effects on ischemic brain damage. All animal studies were approved by the National Institute of Neurological Disorders and Stroke Animal Care and Use Committee. In 98 adult male SHR Ž300–350 g., the left middle cerebral and left common carotid arteries were occluded by a modified tandem occlusion technique w2x under 1–1.5% halothane in 30% O 2 and 70% N2 O. Briefly, the left common carotid artery was ligated and the left middle cerebral artery was exposed and electrocoagu-
) Corresponding author. Stroke Branch, NINDS, NIH, Building 36, Room 4A03, 36 Convent Drive MSC 4128, Bethesda, MD 20892-4128, USA. Fax: q1 Ž301. 402-2769.
lated just distal to the inferior cerebral vein; both vessels were transected. Body temperature was maintained at 37.58C with a heating pad during and 2 h after surgery. The left femoral artery was cannulated for blood sampling and monitoring of blood pressure. 24 h after MCAO, animals were deeply anesthetized with pentobarbital and decapitated. Brains were removed and sequential 2 mm coronal brain sections were incubated at 378C in 2% 2,3,5-triphenyltetrazolium chloride ŽTTC. w9x. The total area of each coronal brain section and the lesion area were image-analyzed and lesion volumes were calculated by integration of the section areas. All analyses were performed in a blinded fashion. The time course for the effects on ischemic brain damage of LPS administered as a single dose was investigated. LPS from Escherichia coli 0111:B4 phenol extract ŽSigma, St. Louis, MO. was dissolved in sterile, pyrogenfree 0.9% Žwrv. NaCl and injected i.v. Ž0.9 mgrkg. 1, 2, 3, 4 or 7 days prior to MCAO. This optimal dose was selected from a series of preliminary dose-finding studies. Control animals were injected with the same volume of sterile, pyrogen-free 0.9% saline at the same time points. In separate experiments, the effect of systemic injection of LPS combined with either IL-1ra or TNFbp on ischemic brain damage was investigated. IL-1ra, a gift from Amgen ŽBoulder, CO., was dissolved in 10 mM sodium citrate
0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 3 8 3 - 2
K. Tasaki et al.r Brain Research 748 (1997) 267–270
268
buffer, pH 6.8, with 150 mM NaCl, 0.5 mM EDTA and 0.1% Tween-80 and injected s.c. 100 mgrkg immediately before and 4 h after LPS injection Ž n s 4.. In another group of animals, human recombinant type I soluble TNF receptor dimer conjugated to polyethylene glycol to enhance its activity, TNFbp, a gift from Amgen ŽBoulder, CO., was dissolved in 10 mM sodium phosphate, 20 mM sodium chloride, pH 6.5 and injected i.v. 2 mgrkg just before LPS injection Ž n s 5.. Control animals for both groups were injected with LPS in combination with an appropriate volume of each vehicle Ž n s 4 and n s 5, respectively.. MCAO was performed 3 days after the LPS q IL-1ra, LPS q TNFbp or LPS q vehicle injection and the infarct volume was measured 24 h post-MCAO by TTC staining. Four subsequent animals received only TNFbp Ž2 mgrkg i.v.. 72 h before MCAO. Serum TNF-a levels were measured by L929 cell lysis bioassay w8x 2 h after LPS q TNFbp or LPS q vehicle injection. Data are presented as mean " S.D. LPS vs. Saline comparisons at each time point and comparison of LPS q IL-1ra vehicle with LPS q IL-1ra were analyzed by independent groups Student’s t-test. The Welch approximation was used when group variances were heterogenous. The TNFbp study was analyzed by one-way ANOVA and pair-wise group comparisons were made with a Bonferroni correction. Student’s t-test was also used to compare the two serum TNF-a levels. Body temperature was not significantly altered after LPS injection and remained - 398C in all animals. Complete blood cell counts revealed no statistically significant differences between the LPS-injected groups and their respective control groups. There were no significant differences in the physiologic variables in any of the LPS- or vehicle-pre-treated groups at any of the 5 time points ŽTable 1.. Similarly, there were no differences in physiologic variables in the blocking studies with LPS q IL-1ra, LPS q TNFbp or their respective controls.
Fig. 1. Infarct volumes: LPS Ž0.9 mgrkg. or saline i.v. at several times before MCAO. ) P - 0.05; ) ) P - 0.02.
Pre-treatment with LPS at 2, 3 or 4 days prior to MCAO reduced the volume of the ischemic damage by 22.8% Ž P - 0.05., 25.9% Ž P - 0.02. and 20.5% Ž P 0.02., respectively. Infarct volumes expressed in mm3 in the 5 groups that constitute the time-course experiments were: saliner1 day 162 " 22, LPSr1 day 138 " 30, saliner2 days 163 " 35, LPSr2 days 122 " 23, saliner3 days 162 " 37, LPSr3 days 116 " 26, saliner4 days 163 " 12, LPSr4 days 128 " 27, saliner7 days 165 " 11, LPSr7 days 163 " 25 ŽFig. 1.. In the blocking studies, ischemic lesion volume was not significantly different between the LPS q IL-1ra-treated group Ž109 " 48 mm3 . and the control group Ž118 " 42 mm3 .. The infarct volume produced by LPS q TNFbp treatment Ž174 " 24 mm3 . was significantly larger than that of its control group Ž119 " 23 mm3, P - 0.03. ŽFig. 2. and was essentially the same volume as that produced by saline pre-treatment. TNFbp pre-treatment alone Ž131 " 19 mm3 . did not increase in-
Table 1 Physiologic variables at occlusion of the middle cerebral artery Time of LPS administration Žpre-MCAO. 1 day LPS Ž n s 8. Saline Ž n s 8. 2 days LPS Ž n s 8. Saline Ž n s 8. 3 days LPS Ž n s 8. Saline Ž n s 8. 4 days LPS Ž n s 8. Saline Ž n s 8. 7 days LPS Ž n s 8. Saline Ž n s 8.
pO 2 Žmm Hg.
pCO 2 Žmm Hg.
pH
Hematocrit Ž%.
Glucose Žmgr100 ml.
MABP Žmm Hg.
155 " 10 136 " 21
39 " 3 39 " 2
7.38 " 0.01 7.42 " 0.02
41 " 2 42 " 1
123 " 12 130 " 11
130 " 12 142 " 7
128 " 38 120 " 16
44 " 2 41 " 4
7.38 " 0.02 7.40 " 0.01
47 " 3 43 " 3
123 " 12 126 " 16
129 " 12 136 " 12
122 " 19 120 " 10
40 " 4 42 " 4
7.40 " 0.01 7.40 " 0.02
44 " 4 46 " 3
121 " 17 130 " 27
132 " 9 138 " 8
142 " 15 133 " 15
41 " 5 38 " 2
7.40 " 0.02 7.41 " 0.02
39 " 2 40 " 4
146 " 17 144 " 10
129 " 10 141 " 11
136 " 15 132 " 22
40 " 2 38 " 5
7.40 " 0.02 7.40 " 0.02
38 " 2 42 " 3
122 " 13 124 " 11
142 " 7 148 " 9
K. Tasaki et al.r Brain Research 748 (1997) 267–270
269
Fig. 2. Infarct volumes: ŽA. MCAO 3 days after LPS q vehicle or LPS q IL-1ra injections; ŽB. MCAO 3 days after LPS q vehicle, LPS q TNFbp, TNFbp only, or saline only injection. ) P - 0.03.
farct volume compared to saline pre-treatment Ž162 " 37 mm3 .. Thus, administration of TNFbp completely nullified the protective effect of LPS pre-treatment. Serum TNF-a levels were measured in an LPS q vehicle group Ž307, 200 " 253, 599 Urml, n s 4. and TNFbp injection coincident with LPS suppressed the serum TNF-a activity Ž80 " 0 Urml, n s 4, P - 0.05.. The present findings demonstrate that systemic LPS administration 48–96 h prior to MCAO induces resistance to subsequent ischemic neuronal damage in SHR. TNFbp, which was demonstrated to greatly inhibit the bioactivity of TNF-a after LPS administration, nullified this effect, indicating that pre-ischemic elevation of TNF-a activity by the LPS is necessary to induce the state of tolerance to subsequent ischemia. Although the LPS was systemically administered and its effects on the cerebral vasculature and circulating cells may account for much of the induced tolerance, systemic LPS also has central actions and can induce TNF-a production by microglia and astrocytes. This raises the possibility that brain parenchymal cells participate in the development of tolerance in this model w1,7,11x. TNF-a pre-treatment has been observed to reduce myocardial damage during perfusion arrest in a Langendorff preparation w5x and lethal decompression sickness in SHR w4x. This is the first demonstration that pre-ischemic administration of LPS induces tolerance to focal brain ischemia. IL-1ra was administered in a dose that has been demonstrated to be neuroprotective during focal brain ischemia in rats w6x. The half-life of IL-1ra is f 6 h after i.p. injection w12x. The biologic activity of the drug in our experimental paradigm was, therefore, probably no longer than 10–12 h after LPS adminmistration. It is possible that continued administration of IL-1ra for several days to more closely match the duration of action of the TNFbp ŽT1r2 24–36 h. w13x would also have demonstrated some attenuation of LPS tolerance induction and, thus, implicated IL-1 in addition to TNF-a as a mediator of this tolerance.
Since our work suggests that tolerance to brain ischemia proceeds from activation of TNF-a receptors, this should greatly facilitate characterization of the intracellular signaling pathways that regulate the tolerant state. This work implicates the p55 and p75 TNF receptors w18x. Activation of TNF receptors leading to production of sphingomyelinbased second messengers and activation of stress-activated protein kinases ŽSPAKs. are, therefore, a potentially relevant series of intracellular responses w10x. Nuclear translocation of NF-k B may also be involved in the stress-induced tolerance observed in this model w3x. Intracellular signaling pathways that originate from TNF-a receptors merit further attention as candidate mechanisms for the regulation of tolerance to ischemia. Acknowledgements We are grateful to Dr. Anna-Leena Siren ´ for measurement of serum TNF-a and to Karen Pettigrew for expert statistical consultation. References w1x Breder, C.D., Hazuka, C., Ghayur, T., Klug, C., Huginin, M., Yasuda, K., Teng, M. and Saper, C.B., Regional induction of tumor necrosis factor a expression in the mouse brain after systemic lipopolysaccharide administration, Proc. Natl. Acad. Sci. USA, 91 Ž1994. 11393–11397. w2x Brint, S., Jacewicz, M., Kiessling, M., Tanabe, J. and Pulsinelli, W., Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries, J. Cereb. Blood Flow Metab., 8 Ž1988. 474–485. w3x Claudio, E., Segade, F., Wrobel, K., Ramos, S., Bravo, R. and Lazo, P.S., Molecular mechanisms of TNF-a cytotoxicity: activation of NF-k B and nuclear translocation, Exp. Cell Res., 224 Ž1996. 63–71. w4x Dutka, A.J. and Broome, J. Spontaneously hypertensive rats ŽSHR. tolerant to endotoxin do not develop decompression illness, Undersea Hyperbaric Med., 21 ŽSuppl.. Ž1994. 92–93. w5x Eddy, L.J., Goeddel, D.V. and Wong, G.H.W., Tumor necrosis factor-a pretreatment is protective in a rat model of myocardial
270
w6x
w7x
w8x
w9x
w10x
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K. Tasaki et al.r Brain Research 748 (1997) 267–270 ischemia-reperfusion injury, Biochem. Biophys. Res. Commun., 184 Ž1992. 1056–1059. Garcia, J.H., Liu, K.-F. and Relton, J.K., Interleukin-1 receptor antagonist decreases the number of necrotic neurons in rats with middle cerebral artery occlusion, Am. J. Pathol., 147 Ž1995. 1477– 1486. Gutierrez, E.G., Banks, W.A. and Kastin, A.J., Murine tumor necrosis factor alpha is transported from blood to brain in the mouse, J. Neuroimmunol., 47 Ž1993. 169–176. Hogan, M.M. and Vogel, S.N., Production of tumor necrosis factor by rIFN-g-primed C3HrHeJ ŽLPSd. macrophages requires the presence of lipid A-associated proteins, J. Immunol., 141 Ž1988. 4196– 4202. Isayama, K., Pitts, L.H. and Nishimura, M.C., Evaluation of 2,3,5triphenyltetrazolium chloride staining to delineate rat brain infarcts, Stroke, 22 Ž1991. 1394–1398. Kyriakis, J.M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E.A., Ahmad, M.F., Avruch, J. and Woodgett, J.R., The stress-activated protein kinase subfamily of c-jun kinases, Nature, 369 Ž1994. 156–160. Laye, ´ S., Parnet, P., Goujon, E. and Dantzer, R., Peripheral administration of lipopolysaccharide induces the expression of cytokine transcripts in the brain and pituitary of mice, Mol. Brain. Res., 27 Ž1994. 157–162.
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