Interleukin-6 prevents ischemia-induced learning disability and neuronal and synaptic loss in gerbils

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Neumscience Letters 204(1996)109-112

NEUROSCHCE LETTEBS

Interleukin-6 prevents ischemia-induced learning disability and neuronal and synaptic loss in gerbils Seiji Matsuda a,*, Tong-Chun Wen a, Fumio Moritaa, Hiroki Otsuka a, Keiji Igase a, Hiroyuki Yoshimura b, Masahiro Sakanaka. aDepartment of Anatomy, Ehime University School of Medicine, Shigenobu, Ehime 791-02, Japan bCentral Research Laboratory, Ehime University School of Medicine, Shigenobu, Ehime 791-02, Japan Received 21 June 1995; revised version received 27 December 1995; accepted 27 December 1995

Abstract

Interleukin-6 (IL-6) has been shown to have potent neurotrophic activity on peripheral and central neurons in vitro. However, it remains to be determined whether or not IL-6 rescues hippocampal CAI neurons from lethal ischemia and prevents ischemia-induced learning disability. In the present in vivo study, we infused IL-6 continuously for 7 days into the lateral ventricle of gerbil starting 2 h before 3-min forebrain iscbemia. IL-6 infusion prevented the occurrence of ischemia-induced learning disability in a dose-dependent manner as revealed by a step-down passive avoidance task. Subsequent light and electron microscopic examinations showed that pyramidal neurons in the CA1 region of the hippocampus as well as synapses within the strata moleculare, radiatum and oriens of the region were significantly more numerous in gerbils infused with IL-6 than in those receiving vehicle infusion. These findings suggest that IL-6 has atrophic effect on ischemic hippocampal neurons.

Keywords: Interleukin-6; Brain ischemia; Passive avoidance task; Delayed neuronal death; Synapse; Gerbil

Cerebral ischemia induces various degrees of brain damage depending on :its intensity and duration. CA1 pyramidal cells in the hippocampus of Mongolian gerbils are selectively vulnerable to transient ischemic insult, and ischemia of 3-5 min duration causes severe neuronal damage to the CA1 neurons which start dying 2 or 3 days after the restoration of circulation [6,10]. A number of neurotrophic factors including acidic and basic fibroblast growth factors, brain-derived neurotrophic factor, ciliary neurotrophic factor and nerve growth factor have been found to prevent the ischemia-induced degeneration of hippocampal CA1 neurons [12,13,17,18]. Moreover, in ischemic gerbils the response latency time of a step-down passive avoidance task has been shown to correlate well with hippocampal CA1 neuronal density [1,14,17,18]. Besides the above growth factors which were originally isolated from neural tissues, certain cytokines produced in lymphocytes have also been considered as neurotrophic factor candidates. Among them is interleukin-6 * Corresponding author. Tel.: +81 89 9645111, ext. 2054; fax: +81 89 9644362; e-mail: [email protected].

(IL-6) which was first discovered as a B-cell stimulating factor [7]. IL-6 facilitates the differentiation of pheochromocytoma PC12 cells into neuronal cells [15] and is capable of supporting the survival of cultured rat cholinergic and catecholaminergic neurons [5,8]. Moreover, Yamada and Hatanaka [19] reported that IL-6 protects cultured rat hippocampal neurons against cell death induced by glutamate which is, at least in part, responsible for ischemic neuronal damage in the hippocampus [3,4]. These in vitro findings raise the possibility that IL-6 subserves an in vivo neurotrophic function in ischemic brain, but experimental proof in support of this speculation is limited. With a passive avoidance task and subsequent histological analysis, the present study was designed to investigate whether or not IL-6 prevents ischemiainduced learning disability and delayed neuronal loss in gerbils. Male Mongolian gerbils weighing 70-80 g (about 12 weeks of age) were housed communally at constant temperature (22 +_ I°C) with a 12:12 h light-dark cycle, and given food and water ad libitum. The following experiments were conducted in accordance with the Guide

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for Animal Experimentation at Ehime University School of Medicine. The animals were anesthetized with 1.5% halothane in a 4:3 mixture of nitrous oxide and oxygen and placed in a stereotaxic apparatus. An osmotic minipump (Alza Corp., Palo Alto, CA, USA) was implanted subcutaneously into the back of each animal, and a needle connected to the minipump was placed in the left lateral ventricle. Human recombinant IL-6 was purchased from Pepro Tech, Inc. (Rocky Hill, NJ, USA) and dissolved in a vehicle (0.05 M phosphate-buffered saline with 0.1% bovine serum albumin). IL-6 at a dose of 45 or 450 ng/day was infused for 7 days into the lateral ventricles of gerbils in which 3-min forebrain ischemia had been induced; control animals, one group with 3-min ischemia and one group of sham-operated animals, received vehicle infusion (n = 8-10 in each group).Two hours after the onset of infusion, both common carotid arteries were ligated with aneurysm clips for 3 min, during which time the temperature of the brain and rectum was maintained at 37 _+0.2°C [10]. Seven days after forebrain ischemia, the gerbils were examined with a conventional step-down passive avoidance apparatus, as described elsewhere [ 1,14,17,18]. One hour after the passive avoidance experiments, each animal was anesthetized with pentobarbital, and the osmotic minipump was disconnected from the needle that had been placed in the lateral ventricle. Bromophenol blue was injected through the needle to see dye diffusion into the cerebral ventricles. Then the animals were perfused transcardially with 4% paraformaldehyde-2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) and processed for light and electron microscopy [14,18]. Viable neurons along 1 mm linear length of the CA1 field in six serial coronal sections (1.20-1.23 mm posterior to bregma) were counted and the mean number of neurons per section was calculated for each animal. Left-to-right difference in CA1 neuronal density was noted only once in eight cases, regardless of the side of IL-6 infusion. In these animals with left-to-right difference, the mean of both sides of neuronal density was calculated. All cellcounting was done blind with respect to experimental group. An electron micrograph was taken from the central area (15 x 18.75/zm = 280/am 2) of each stratum of the CA1 region where the neuronal inputs and the form of dendritic arborization are different between individual strata [2]. Intact synapses with synaptic vesicles in the presynaptic structure and with normal pre- and postsynaptic elements were counted at a final magnification of 12 000. The blood flow and temperature of the hippocampus prior to or after 3-min ischemia were checked in gerbils with the continuous infusion of IL-6 at a dose of 450 ng/ day or with vehicle infusion into the lateral ventricles (n = 8 in each group). The hippocampal blood flow was de-

termined by the hydrogen clearance method with the use of a digital blood flow meter (MHG-DI; Unique Medical, Tokyo, Japan) under inhalation anesthesia. The hippocampal temperature was monitored with the combination of a temperature-sensitive probe (Mini Mitter Co., Sunriver, USA) inserted into the hippocampus and the Telemetry System receiving signals from the probe (Datascience Int., Minneapolis, USA), while the animals gained free access to food and water except for the periods of minipump implantation and forebrain ischemia. The effect of IL-6 was evaluated by the two-tailed Mann-Whitney U-test. All data were represented as median _+ interquartile range. IL-6 infusion into the lateral ventricle starting 2 h before forebrain ischemia and continuing for 7 days, caused a dose-dependent prolongation in response latency in the step-down passive avoidance task (Fig. 1A). There was a significant difference between 450 ng/day IL-6 and vehicle treatment in ischemic animals (U = 2.5, P < 0.01). The median response latency in ischemic gerbils with 450 ng/ day of IL-6 infusion was close to that in sham-operated animals with vehicle infusion, (450 ng/day IL-6-treated animals, 198 _+ 14.8 s; sham-operated animals, 223 +_ 23.5 s). Subsequent histological examinations revealed that IL-6 treatment rescued dose-dependently many ischemic CA1 neurons that were destined to degenerate without the treatment (Figs. 1B, 2) (45 ng/day IL-6 versus vehicle in ischemic animals: U = 13, P < 0.05; 450 ng/day IL-6 versus vehicle in ischemic animals: U = 1, P < 0.01). The median number of viable CA1 neurons in the gerbils treated with the higher dose of IL-6 was, if not the same

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Fig. 1. Effects of IL-6 on leaming disability (A) and neuronal density of the hippocampal CAI region (B) in gerbils with 3-min ischemia. Treatment of ischemic gerbils with IL-6 (450 ng/day) over a 7-day period prolonged significantly response latency time in the passive avoidance task, in comparison with the vehicle-treated ischemic gerbils (A). Neurons in the hippocampal CA1 region of ischemic gerbils with vehicle infusion were less numerous than those of sham-operated (sham-ope) animals, and the decrease in neuron number in the ischemic hippocampus was prevented significantly in a dose-dependent manner when IL-6 was infused into the lateral ventricle for 7 days (B). Each value represents median ± interquartile range (n = 8-10). *P < 0.05, **P < 0.01, significantly different from the ischemic group with vehicle infusion. Open columns, sham operation; closed columns, ischemia.

S. Matsuda et al. I Neuroscience Letters 204 (1996) 109-112

as, close to that in the sham-operated animals (450 ng/day IL-6-treated animals, 202 _ 8.3 cells/mm; sham-operated animals, 245 __.6.5 cells/lama). Under electron microscopy, degenerating postsynaptic elements of CA1 neuron origin exhibited relatively high electron density (Fig. 3). In line with the results of the passive avoidance task and light microscopic observations, electron microscopy showed that intact synapses within the stratum moleculare, stratum radiatum and stratum oriens of the hippocampal CA1 region were more numerous in IL-6-treated than in vehicle-treated ischemic gerbils (Fig. 3) (45 ng/day IL-6 versus vehicle in the individual strata of ischernic animals: U = 0, P < 0.01; U = 4, P < 0.01; U = 1, P < 0.01; 450 ng/day IL-6 versus vehicle in the individual strata of ischemic animals: U = 0, P < 0.01; U = 2.5, P < 0.01; U = 7, P < 0.01). There was no significant difference in hippocampal blood flow and hippocampal temperature between IL-6infused and vehicle-treated gerbils before and 5-120 min after forebrain ischemia. The mechanisms by which IL-6 rescues hippocampal neurons from lethal isclhemic damage remain to be determined. Since intraventricular IL-6 infusion did not affect hippocampal blood flow and brain temperature, it is not unlikely that IL-6 acts directly on the brain. Indeed, the mRNA of IL-6 receptor is present in the pyramidal cells within the CA1--CA4 fields of hippocampus [16]. Moreover, IL-6 immunoreactivity is increased in astrocytes from ischemic regions of gerbil brain [9]. These findings may suggest that IL-6 of neuroglial origin acts

Fig. 2. Photomicrographs of the hippocampal CAI region of gerbils with or without 3-min ischelrda. (A) Sham-operated animal with vehicle infusion. (B) Isehemic animal with vehicle infusion. (C) Ischemic animal with IL-6 (45 rig/day) infusion. (D) Ischemic animal with 1L-6 (450 rig/day) infusion. Note that many hippocampal CAI pyramidal cells are rescued by the treatment with IL-6. All sections are stained with cresyl violet. Bar = 50/~m.

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Fig. 3. (A,B) Electron micrographs showing intact synapses (arrows) and degenerated synapses (arrowheads) in the ischemic hippocampus treated with either vehicle (A) or IL-6 (B). Bar = 1/~m. (C) Effect of IL-6 on the number of synapses in three strata of the hippoeampal CA1 region. Synapses in the strata moleculare, radiatum and oriens of the hippocampal CA1 region of ischemic gerbils with vehicle infusion were less numerous than those in the three strata of sham-operated (sham-ope) animals, and the decrease in synapse number in the ischemic hippoeampus was prevented significantly when IL-6 (45 ng/day or 450 ng/day) was infused into the lateral ventricle for 7 days. Each value represents median ± interquartile range (n = 8-10). **P < 0.01, significantly different from the ischemic group with vehicle infusion. Open columns, sham operation; closed columns, ischemia.

directly on ischemic hippocampal neurons by binding to the cell surface receptor in a paracrine manner. Maeda et al. [9] also demonstrated that in ischemic gerbils IL-6 activity in the hippocampus vulnerable to ischemia is lower than that in the cerebral cortex resistant to ischemia; so endogenous IL-6, even though increasing in local content in response to ischemic insult, may not have been able to rescue many CA1 neurons in the hippocampus of 3-min ischemic gerbils treated with vehicle. In contrast, exogenous IL-6 infused continuously into the cerebral ventricle in this study is thought to have prevented delayed neuronal death by acting on ischemic CA1 neurons in need of exogenous as well as endogenous IL-6. Like IL-6, ciliary neurotrophic factor (CNTF) has been shown to prevent ischemic neuronal loss and learning disability [18]. Since CNTF shares the use of a signaltransducing receptor component, gpl30, with IL-6 [11], tyrosine phosphorylation of gpl30 in response to IL-6 or CNTF treatment may be a common signal transduction in favor of neuronal survival during brain ischemia. In conclusion, the present study first demonstrated that IL-6, when continuously infused into the lateral ventricles of gerbils with 3-min forebrain ischemia, prevents the

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occurrence of learning disability and hippocampal neuron loss. Dr. Tong-Chun Wen is on leave of absence from Taishan Medical College, People's Republic of China. The authors wish to express warm appreciation to President Chongrui Jia of Taishan Medical College for encouraging us throughout this work. We are grateful to Miss Mika Fujimoto for her secretarial assistance. This work was supported, in part, by Uehara Memorial Foundation. [1] Araki, H., Nojiri, M., Kawashima, K., Kimura, M. and Aihara, H., Behavioral, electroencephalographic and histopathological studies on Mongolian gerbils with occluded common carotid arteries, Physiol. Behav., 38 (1986) 89-94. [2] Bayer, S.A., Hippocampal region. In G. Paxinos (Ed.), The Rat Nervous System, Vol. 1, Academic Press, Sydney, 1985, pp. 335352. [3] Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extraceilular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1984) 1369-1374. [4] Benveniste, H., Jorgenson, M.B., Sandberg, M., Christensen, T., Hagberg, H. and Diemer, N.H., Ischemic damage in hippocampal CA1 is dependent on glutamate release and intact innervation from CA3, J. Cereb. Blood Flow Metab., 9 (1989) 629-639. [5] Hama, T., Miyamoto, M., Tsukui, H., Nishio, C. and Hatanaka, H., Interleukin-6 as a neurotrophic factor for promoting the survival of cultured basal forebrain cholinergic neurons from postnatal rats, Neurosci. Lett., 104 (1989) 340-344. [6] Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 237 (1982) 57-69. [7] Kishimoto, T., The biology of interleukin-6, Blood, 74 (1989) 110. [8] Kushima, Y., Hama T. and Hatanaka H., Interleukin-6 as a neurotrophic factor for promoting the survival of cultured catecholaminergic neurons in a chemically defined medium, Neurosci. Res., 13 (1992) 267-280. [9] Maeda, Y., Matsumoto, M., Hori, O., Kuwabara, K., Ogawa, S., Yan, SD., Ohtsuki, T., Kinoshita, T., Kamada, T. and Stem, DM.,

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19]

Hypoxia/reoxygenation-mediated induction of astrocyte interleukin 6: a paracrine mechanism potentially enhancing neuron survival, J. Exp. Med., 180 (1994) 2297-2308. Mitani, A., Andou, Y., Masuda, S. and Kataoka, K., Transient forebrain ischemia of three-minute duration consistently induces severe neuronal damage in field CA! of the hippocampus in the normothermic gerbil, Neurosci. Lett., 131 (1991) 171-174. Murakami, M., Hibi, M., Nakagawa, N., Nakagawa, T., Yasukawa, K., Yamanashi, K., Taga, T. and Kishimoto, T., IL-6 induced homodimerization of gpl30 and associated activation of a tyrosine kinase, Science, 260 (1993) 1808-1810. Nakata, N., Kato, H. and Kogure, K., Protective effects of basic fibroblast growth factor against hippocampal neuronal damage following cerebral ischemia in the gerbil, Brain Res., 605 (1993) 354-356. Sakaki, K., Oomura, Y., Suzuki, K., Hanai, K. and Yagi, H., Acidic fibroblast growth factor prevents death of hippocampal CAI pyramidal cells following ischemia, Neurochem. Int., 21 (1992) 397-402. Sano, A., Matsuda, S., Wen, T.-C., Kotani, Y., Kondoh, K., Ueno, S., Kakimoto, Y., Yoshimura, H. and Sakanaka, M., Protection by prosaposin against ischemia-induced learning disability and neuronal loss, Biochem. Biophys. Res. Commun., 204 (1994) 9941000. Sato, T., Nakamura, S., Taga, T., Matsuda, T., Hirano, T., Kishimoto, T. and Kaziro, Y.. Induction of neuronal differentiation of PC12 cells by B cell stimulatory factor 2/interleukin 6, Mol. Cell Biol., 8 (1988) 3346-3546. SchObitz, B., Voorhuis, D.A.M., and De Kloet, E.R., Localization of interleukin 6 mRNA and interleukin 6 receptor mRNA in rat brain, Neurosci. Lett., 136 (1992) 189-192. Wen, T.-C., Matsuda, S., Yoshimura, H., Aburaya, J., Kushihata, F. and Sakanaka, M., Protective effect of basic fibroblast growth factor-heparin and neurotoxic effect of platelet factor 4 on ischemic neuronal loss and learning disability in gerbils, Neuroscience, 65 (1995) 513-521. Wen, T.-C., Matsuda, S., Yosbimura, H., Kawab¢, T. and Sakanaka, M., Ciliary neurotrophic factor prevents ischemia-induced learning disability and neuronal loss in gerbils, Neurosci. I.~tt., 191 (1995) 55-58. Yamada, M. and Hatanal(a, H., Interleukin-6 protects cultured rat hippocampal neurons against glutamate-induced cell death, Brain Res., 643 (1994) 173-180.