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Ciliary neurotrophic factor prevents ischemia-induced learning disability and neuronal loss in gerbils
ELSEVIER
Neuroscience Letters 191 (1995) 55-58
Ciliary neurotrophic factor prevents ischemia-induced learning disability and neuronal loss in gerbils Tong-Chun Wena, Sejii Matsudaa,*, Hiroyuki Yoshimurab, Tadao Kawabea, Masahiro Sakanakaa aDepartrnent o f A n a t o m y , E h i m e Universiry S c h o o l o f M e d i c i n e , S h i g e n o b u , E h i m e 7 9 1 - 0 2 , J a p a n bCentral R e s e a r c h L a b o r a t o r y , E h i m e U n i v e r s i t y S c h o o l o f M e d i c i n e , S h i g e n o b u , E h i m e 7 9 1 - 0 2 , J a p a n
Received 16 December 1994; revised version received 13 April 1995; accepted 13 April 1995
Abstract Ciliary neurotrophic factor (CNTF) has been shown to have potent neurotrophic activity on peripheral and central neurons in vitro and in vivo. However, it remains to be determined whether or not CNTF rescues hippocampal CA1 neurons from lethal ischemia and prevents ischemia-induced learning disability. In the present in vivo study, we infused CNTF continuously for 7 days into the lateral ventricle of gerbil starting 2 h before 3-min forebrain ischemia. CNTF infusion prevented the occurrence of ischemia-induced learning disability in a dose-dependent manner as revealed by the 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, lacunosum/radiatum and oriens of the region were significantly more numerous in gerbils infused with CNTF than in those receiving vehicle infusion. These findings suggest that CNTF has a trophic effect on ischemic hippocampal neurons. Keywords:
Ciliary neurotrophic factor; 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 &hernia 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 [8,10]. A number of neurotrophic factors have been found to prevent the ischemia-induced degeneration of hippocampal CA1 neurons. Acidic and basic fibroblast growth factors have been shown to protect hippocampal CA1 pyramidal neurons against delayed neuronal death following transient forebrain ischemia in gerbils [ 11,131. Shigeno et al. [ 161 demonstrated that nerve growth factor reduces the neuronal loss of hippocampal CA1 neurons in ischemic gerbils. Another type of trophic factor, ciliary neurotrophic factor (CNTF), was initially identified, purified and molecularly cloned because of its ability to support the in vitro survival of parasympathetic neurons from the chick * Corresponding author, Tel.: +81 899 64 5111, ext. 2054; Fax: +81 899 64 4362.
0304-?940/95/$09.50 SSDI 0304.3940(9X)
0 1995 Elsevier Science Ireland Ltd. All rights reserved I 1574-D
ciliary ganglion [2]. Subsequent in vitro and in vivo studies have revealed that CNTF supports neurons from several brain regions, including the hippocampus [4,6,15]. In addition, CNTF has been shown to promote the development of neurons and glial cells [5] and the differentiation of 02A progenitor cells to type 2 astrocytes [9]. However, little is known of the neurotrophic action of CNTF on ischemic hippocampal neurons. The present study was designed to investigate, with passive avoidance task and subsequent morphological analyses, whether or not CNTF rescues hippocampal CA1 neurons from lethal ischemic insult. Male Mongolian gerbils weighing 70-80 g (about 12 weeks of age) were housed communally at constant temperature (22 + 1°C) with a 12:12 h light/dark cycle, and given food and water ad libitum. They were handled once a week for cage cleaning. The following experiments were conducted in accordance with the Guide 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.,
56
T.-C.
Wen et ai. I Neuroscience Letters 191 (1995) 55-58
Palo Alto, CA, USA) was implanted subcutaneously into the back of each animal, and a needle from the minipump was placed in the left lateral ventricle. Human recombinant CNTF was purchased from Genzyme (Cambridge, MA 02139, USA) and dissolved in a vehicle (0.05 M phosphate-buffered saline with 0.1% bovine serum albumin). CNTF at a dose of 50 or 500 rig/day was infused for 7 days into the lateral ventricles of normothermic 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-l 1 in each group). Two hours after the onset of infusion, both common carotid arteries were exposed through a midline incision and separated carefully from the adjacent veins and nerves. The 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, as described elsewhere [101. Seven days after forebrain ischemia, the gerbils were trained in a conventional step-down passive avoidance apparatus which was divided into a safe platform and an experimental chamber with a stainless-steel grid floor [ 11. Training of passive avoidance was carried out 7 days after forebrain ischemia. Each animal was placed initially on the safe platform. When the gerbil stepped down onto the grid floor, it received a foot shock. Although the gerbil went repeatedly up and down between the platform and the grid, it eventually remained on the platform. This training session lasted 300 s. Twenty-four hours later, the gerbil was again placed on the safe platform while the shock generator was turned off, and the response latency, i.e. the time till it stepped down onto the grid floor, was measured. This test session also lasted 300 s [ 14,181. Each animal received only one training session and only one test session. 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 they were perfused transcardially with 4% paraformaidehyde/2.5% glutaraldebyde in 0.1 M phosphate buffer (pH 7.4) for light and electron microscopy, as described elsewhere [ 141. Briefly, a brain region including the dorsal hippocampus from 0.5 to 1.5 mm posterior to bregma was removed and kept in the same fixative overnight at 4°C. Four serial coronal sections 50pm thick at the level 1.0-1.2 mm posterior to bregma were cut with a microslicer for electron microscopy. The remaining dorsal hippocampus was embedded in paraffin, and 5pm serial frontal sections were cut and stained with 0.1% cresyl violet. Viable neurons along 1 mm linear length of the CA1 sub-field in 6 serial coronal sections (1.20-l .23 mm posterior to bregma)
were counted with an image analyzer (Nexus Co. Ltd, Tokyo). The mean number of neurons per section -was calculated for each animal. All cell-counting was done blind with respect to experimental group. Specimens for electron microscopy were dehydrated in ethanol and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate. An electron micrograph was taken from the central area (15pm X 18.75pm = 280pm2) of each stratum. Intact synapses with thick apposed membranes and synaptic vesicles were counted at a final magnification of 12 000. The effect of CNTF was evaluated by the one-tailed Mann-Whitney U-test, which enabled us to compare the CNTF-treated groups with the vehicle-treated ischemic group. The P-value smaller than 0.025 was considered to be significant. All data were represented as median +- interquartile range. CNTF infusion into the lateral ventricle starting 2 h before forebrain ischemia and continuing for 7 days, caused a significant dose-dependent prolongation in response latency in the step-down passive avoidance task (Fig. 1 A) (50 rig/day CNTF versus vehicle in ischemic animals: U = 12.5, P < 0.025; 500 nglday CNTF versus vehicle in ischemic animals: U = 5, P < 0.01). The median response latency in ischemic gerbils with 500 rig/day of CNTF infusion was close to that in sham-operated animals witln vehicle infusion (500 rig/day CNTF treated animals, 225 s 30.5 s; sham-operated animals, 233 * 41.5 s). Subsequent histological examinations revealed that CNTF treatment rescued many ischemic CA1 neurons that were destined to degenerate without the treatment (Figs. lB, 2) (50 ng/ day CNTF versus vehicle in ischemic animals: U = 13, A
Effect of CNTF cm respmse latency * * / T
Effect of CNTF on GA1 neuronal density r - - - - - l
0 shaype vehicle vehicle
50 CNTF(ng/da
shayope 500 vehicle 50 CNTFtnglday) vehicle
Fig. 1. Effects of CNTF on learning disability (A) and neuronal density of the hippocampal CA1 region (B) in gerbils with 3-min ische.xia. Treatment with CNTF over a 7-day period (500ng/day) abolished ischemia-induced learning disability as revealed by the passive avoidance task (A). Neurons in the hippocampaf CA1 region of ischemic gerbils with vehicIe infusion were less numerous than those of shamoperated (sham-ope) animals, and the decrease in neuron number in the ischemic hippocampus was prevented significantly in a dose-dependent manner wben CNTF was infused into the laterai ventricie for 7 days (B). Each value represents median f interquartile range (n = g-11). *P < 0.025, **P < 0.01 significantly different from the ischemic group with vehicle infusion. Open column, sham operation; closed colnmns, ischemia.
T.-C. Wen et al. I Neuroscience Letters 191 (I 995) 55-58
Fig. 2. Photomicrographs of the hippocampal CA1 region of gerbils with or without 3-min ischemia. (A) Sham-operated animal with vehicle infusion. (B) ischemic animal with vehicle infusion. (C) Ischemic animal with CNTF (500 rig/day) infusion. Note that many hippocampal CA1 pyramidal cells are rescued by the treatment with CNTF. All sections are stained with cresyl violet. Bar = 100ym.
P < 0.025; 500 nglday CNTF versus vehicle in ischemic animals: V = 7, P c 0.01). The median number of viable CA1 neurons in the gerbils treated with the higher dose of CNTF was nearly the same as that in the shamoperated animals (500 rig/day CNTF-treated animals, 225 + 17.5 cells/mm; sham-operated animals, 249 + 7.8 cells/mm). In line with the results of the passive avoidance task and light microscopic observations, electron microscopy showed that synapses within the stratum moleculare, stratum lacunosum/radiatum and stratum oriens of the hippocampal CA1 region were more numerous in CNTFtreated than in vehicle-treated ischemic gerbils (Fig. 3) (50 rig/day CNTF versus vehicle in the individual strata of ischemic animals: V = 7, P < 0.01; V = 13, P < 0.01; V = 14.5, P c 0.025; 500 ngfday CNTF versus vehicle in
Fig. 3. Effect of CNTF on the number of synapses in three strata of the hippocampal CA1 region. Synapses in the strata molecuhue, lacunosumkadiatum 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 hippocampus was prevented significantly when CNTF (50 &day or 500 rig/day) was infused into the lateral ventricle for 7 days. Each value represents median k interquartile range (n = 8-11). *P < 0.025, **P < 0.01, significantly different from the ischemic group with vehicle infusion. Open column, sham operation; closed columns, ischemia.
the individual strata of ischemic animals: V = 3, P < 0.01; V = 5, P < 0.01; V = 8, P < 0.01). During forebrain ischemia, brain temperature has been shown to fall to different degrees in individual animals, thereby affecting the number of viable CA1 neurons after ischemia [lo]. To avoid the effect of unstable brain temloss, we kept the brain perature on ischemic neuronal temperature at 37.0 f 0.2”C while clamping the common carotid arteries [ 101. This enabled us to apply the same intensity of ischemic insult to all animals and to evaluate with accuracy the neuroprotective effect of CNTF in gerbils with 3-min ischemia. The mechanism(s) by which CNTF exerts a neuroprotective effect on ischemic hippocampal CA1 neurons is not fully understood, although it is possible that CNTF has neurotrophic activity. Brain ischemia has been shown to induce excessive release of endogenous glutamate which further facilitates calcium influx into the cytoplasm of ischemic neurons by binding to N-methyl-D-aspartate receptor [3,12]. Skaper et al. El71 demonstrated that CNTF raises the threshold of hippocampal pyramidal neuron sensitivity to glutamate toxicity, thereby protecting cultured hippocampal neurons against injury due to energy substrate deprivation and excessive glutamate. It is plausible to ascribe the neurotrophic action of CNTF on ischemic CA1 neurons in this study to the suppression by CNTF of neuronal sensitivity to increased extracellular glutamate during and after ischemia. Recently, a functional CNTF receptor (a-receptor) was found in neurons of the hippocampus [7]. This may indicate that CNTF acts on hippocampal neurons by binding to its receptor on the cell surface. In conclusion, the present study first demonstrated that CNTF, when continuously infused into the lateral ventricles of gerbils with 3-min forebrain ischemia, prevents the occurrence of learning disability based on a passive avoidance task and hippocampal neuron loss.
58
T.-C. Wen et al. I Neuroscience Letters 19P(1995) 55-58
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 the Uehara Memorial Foundation. [l] 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. [Z] Barbin, G., Manthorpe, M. and Varon, S., Purification of the chick eye ciliary neurotrophic factor, J. Neurochem., 43 (1984) 1468-1478. [3] Choi, D.W. and Rothman, S.M., The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death, Annu. Rev. Neurosci., 13 (1990) 171-182. [4] Clatterbuck, R.E., Price, D.L. and Koliatsos, V.E., Ciliary neurotrophic factor prevents retrograde neuronal death in the adult central nervous system, Proc. Natl. Acad. Sci. USA, 90 (1993) 2222-2226. 151 Hughes, S.M., Lillien, L.E., Raff, M.C., Rohrer, H. and Sendtner, M., Ciliary neurotrophic factor induces type-2 astrocyte differentiation in culture, Nature, 335 (1988) 70-73. 161 Ip, N.Y., Li, Y., van de Stadt, I., Panayotatos, N., Alderson, R.F. and Lindsay, R.M., Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures, J. Neurosci., 11 (1991) 31243134. [7] Ip, N.Y., McClain, J., Barrezueta, N.X., Aldrich, T.H., Pan, L., Li, Y., Wiegand, S.J., Friedman, B., Davis, S. and Yancopoulos, G.D., The a-component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development, Neuron, 10 (1993) 89-102. [S] Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 237 (1982) 57-69. [9] Lillien, L.E., Sendtner, M., Rohrer, H., Hughes, SM. and Raff,
DOI
VII
v21 I131
114]
El51 [I61
H71
[I81
M.C., Type-2 astrocyte deveiopment in rat brain cultures is initiated by a CNTF-like protein produced by type-l astrocytes, Neuron, 1 (1988) 485-494. Mitani, A., Andou, Y., Masuda, S. and Kataoka, K., Transient forebrain ischemia of three-minute duration consistently induces severe neuronal damage in field CA1 of the hippocampus in the normothermic gerbil, Neurosci. Lett., 131 (1991) 171-174. 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. Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemic brain damage, Ann. Neurol., 19 (1986) 105-111. Sakaki, K., Oomura, Y., Suzuki, K., Hanai, K. and Yagi, II., Acidic tibroblast growth factor prevents death of hip~c~pal CA1 pyramidal cells following ischemia, Neurochem. Int., 21 (1992) 397-402. &no, 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. Sendtner, M., Kreutzberg, G.W. and Thoenen, H., Ciliary neurotrophic factor prevents the degeneration of motor neurons after axotomy, Nature, 345 (1990) 440-441. Shigeno, T., Mima, T., Takakura, K., Graham, D.I., Kate, G., Hashimoto, Y. and Furukawa, S., Amelioration of delayed neuronal death in the hippocampus by nerve growth factor, J. Neurosci., 11 (1991) 2914-2919. Skaper, SD., Negro, A., Dal Toso, R, and Facei, L., Recombinant human ciliary neurotrophic factor alters the threshold of hippocampal pyramidal neuron sensitivity to excitotoxin damage: synergistic effects of monosialogangliosides, J. Neurosci. Res., 33 (1992) 330-337. Wen, T.-C., Matsuda, S., Yoshimura, H., Aburaya, J., Kushihata, F. and Sakanaka, M., Protective effect of basic flbroblast growth factor-heparin and neurotoxic effect of platelet factor 4 on ischemic neuronal loss and learning disability in gerbils, Neuroscience, 65 (1995) 513-521.
Neuroscience Letters 191 (1995) 55-58
Ciliary neurotrophic factor prevents ischemia-induced learning disability and neuronal loss in gerbils Tong-Chun Wena, Sejii Matsudaa,*, Hiroyuki Yoshimurab, Tadao Kawabea, Masahiro Sakanakaa aDepartrnent o f A n a t o m y , E h i m e Universiry S c h o o l o f M e d i c i n e , S h i g e n o b u , E h i m e 7 9 1 - 0 2 , J a p a n bCentral R e s e a r c h L a b o r a t o r y , E h i m e U n i v e r s i t y S c h o o l o f M e d i c i n e , S h i g e n o b u , E h i m e 7 9 1 - 0 2 , J a p a n
Received 16 December 1994; revised version received 13 April 1995; accepted 13 April 1995
Abstract Ciliary neurotrophic factor (CNTF) has been shown to have potent neurotrophic activity on peripheral and central neurons in vitro and in vivo. However, it remains to be determined whether or not CNTF rescues hippocampal CA1 neurons from lethal ischemia and prevents ischemia-induced learning disability. In the present in vivo study, we infused CNTF continuously for 7 days into the lateral ventricle of gerbil starting 2 h before 3-min forebrain ischemia. CNTF infusion prevented the occurrence of ischemia-induced learning disability in a dose-dependent manner as revealed by the 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, lacunosum/radiatum and oriens of the region were significantly more numerous in gerbils infused with CNTF than in those receiving vehicle infusion. These findings suggest that CNTF has a trophic effect on ischemic hippocampal neurons. Keywords:
Ciliary neurotrophic factor; 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 &hernia 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 [8,10]. A number of neurotrophic factors have been found to prevent the ischemia-induced degeneration of hippocampal CA1 neurons. Acidic and basic fibroblast growth factors have been shown to protect hippocampal CA1 pyramidal neurons against delayed neuronal death following transient forebrain ischemia in gerbils [ 11,131. Shigeno et al. [ 161 demonstrated that nerve growth factor reduces the neuronal loss of hippocampal CA1 neurons in ischemic gerbils. Another type of trophic factor, ciliary neurotrophic factor (CNTF), was initially identified, purified and molecularly cloned because of its ability to support the in vitro survival of parasympathetic neurons from the chick * Corresponding author, Tel.: +81 899 64 5111, ext. 2054; Fax: +81 899 64 4362.
0304-?940/95/$09.50 SSDI 0304.3940(9X)
0 1995 Elsevier Science Ireland Ltd. All rights reserved I 1574-D
ciliary ganglion [2]. Subsequent in vitro and in vivo studies have revealed that CNTF supports neurons from several brain regions, including the hippocampus [4,6,15]. In addition, CNTF has been shown to promote the development of neurons and glial cells [5] and the differentiation of 02A progenitor cells to type 2 astrocytes [9]. However, little is known of the neurotrophic action of CNTF on ischemic hippocampal neurons. The present study was designed to investigate, with passive avoidance task and subsequent morphological analyses, whether or not CNTF rescues hippocampal CA1 neurons from lethal ischemic insult. Male Mongolian gerbils weighing 70-80 g (about 12 weeks of age) were housed communally at constant temperature (22 + 1°C) with a 12:12 h light/dark cycle, and given food and water ad libitum. They were handled once a week for cage cleaning. The following experiments were conducted in accordance with the Guide 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.,
56
T.-C.
Wen et ai. I Neuroscience Letters 191 (1995) 55-58
Palo Alto, CA, USA) was implanted subcutaneously into the back of each animal, and a needle from the minipump was placed in the left lateral ventricle. Human recombinant CNTF was purchased from Genzyme (Cambridge, MA 02139, USA) and dissolved in a vehicle (0.05 M phosphate-buffered saline with 0.1% bovine serum albumin). CNTF at a dose of 50 or 500 rig/day was infused for 7 days into the lateral ventricles of normothermic 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-l 1 in each group). Two hours after the onset of infusion, both common carotid arteries were exposed through a midline incision and separated carefully from the adjacent veins and nerves. The 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, as described elsewhere [101. Seven days after forebrain ischemia, the gerbils were trained in a conventional step-down passive avoidance apparatus which was divided into a safe platform and an experimental chamber with a stainless-steel grid floor [ 11. Training of passive avoidance was carried out 7 days after forebrain ischemia. Each animal was placed initially on the safe platform. When the gerbil stepped down onto the grid floor, it received a foot shock. Although the gerbil went repeatedly up and down between the platform and the grid, it eventually remained on the platform. This training session lasted 300 s. Twenty-four hours later, the gerbil was again placed on the safe platform while the shock generator was turned off, and the response latency, i.e. the time till it stepped down onto the grid floor, was measured. This test session also lasted 300 s [ 14,181. Each animal received only one training session and only one test session. 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 they were perfused transcardially with 4% paraformaidehyde/2.5% glutaraldebyde in 0.1 M phosphate buffer (pH 7.4) for light and electron microscopy, as described elsewhere [ 141. Briefly, a brain region including the dorsal hippocampus from 0.5 to 1.5 mm posterior to bregma was removed and kept in the same fixative overnight at 4°C. Four serial coronal sections 50pm thick at the level 1.0-1.2 mm posterior to bregma were cut with a microslicer for electron microscopy. The remaining dorsal hippocampus was embedded in paraffin, and 5pm serial frontal sections were cut and stained with 0.1% cresyl violet. Viable neurons along 1 mm linear length of the CA1 sub-field in 6 serial coronal sections (1.20-l .23 mm posterior to bregma)
were counted with an image analyzer (Nexus Co. Ltd, Tokyo). The mean number of neurons per section -was calculated for each animal. All cell-counting was done blind with respect to experimental group. Specimens for electron microscopy were dehydrated in ethanol and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate. An electron micrograph was taken from the central area (15pm X 18.75pm = 280pm2) of each stratum. Intact synapses with thick apposed membranes and synaptic vesicles were counted at a final magnification of 12 000. The effect of CNTF was evaluated by the one-tailed Mann-Whitney U-test, which enabled us to compare the CNTF-treated groups with the vehicle-treated ischemic group. The P-value smaller than 0.025 was considered to be significant. All data were represented as median +- interquartile range. CNTF infusion into the lateral ventricle starting 2 h before forebrain ischemia and continuing for 7 days, caused a significant dose-dependent prolongation in response latency in the step-down passive avoidance task (Fig. 1 A) (50 rig/day CNTF versus vehicle in ischemic animals: U = 12.5, P < 0.025; 500 nglday CNTF versus vehicle in ischemic animals: U = 5, P < 0.01). The median response latency in ischemic gerbils with 500 rig/day of CNTF infusion was close to that in sham-operated animals witln vehicle infusion (500 rig/day CNTF treated animals, 225 s 30.5 s; sham-operated animals, 233 * 41.5 s). Subsequent histological examinations revealed that CNTF treatment rescued many ischemic CA1 neurons that were destined to degenerate without the treatment (Figs. lB, 2) (50 ng/ day CNTF versus vehicle in ischemic animals: U = 13, A
Effect of CNTF cm respmse latency * * / T
Effect of CNTF on GA1 neuronal density r - - - - - l
0 shaype vehicle vehicle
50 CNTF(ng/da
shayope 500 vehicle 50 CNTFtnglday) vehicle
Fig. 1. Effects of CNTF on learning disability (A) and neuronal density of the hippocampal CA1 region (B) in gerbils with 3-min ische.xia. Treatment with CNTF over a 7-day period (500ng/day) abolished ischemia-induced learning disability as revealed by the passive avoidance task (A). Neurons in the hippocampaf CA1 region of ischemic gerbils with vehicIe infusion were less numerous than those of shamoperated (sham-ope) animals, and the decrease in neuron number in the ischemic hippocampus was prevented significantly in a dose-dependent manner wben CNTF was infused into the laterai ventricie for 7 days (B). Each value represents median f interquartile range (n = g-11). *P < 0.025, **P < 0.01 significantly different from the ischemic group with vehicle infusion. Open column, sham operation; closed colnmns, ischemia.
T.-C. Wen et al. I Neuroscience Letters 191 (I 995) 55-58
Fig. 2. Photomicrographs of the hippocampal CA1 region of gerbils with or without 3-min ischemia. (A) Sham-operated animal with vehicle infusion. (B) ischemic animal with vehicle infusion. (C) Ischemic animal with CNTF (500 rig/day) infusion. Note that many hippocampal CA1 pyramidal cells are rescued by the treatment with CNTF. All sections are stained with cresyl violet. Bar = 100ym.
P < 0.025; 500 nglday CNTF versus vehicle in ischemic animals: V = 7, P c 0.01). The median number of viable CA1 neurons in the gerbils treated with the higher dose of CNTF was nearly the same as that in the shamoperated animals (500 rig/day CNTF-treated animals, 225 + 17.5 cells/mm; sham-operated animals, 249 + 7.8 cells/mm). In line with the results of the passive avoidance task and light microscopic observations, electron microscopy showed that synapses within the stratum moleculare, stratum lacunosum/radiatum and stratum oriens of the hippocampal CA1 region were more numerous in CNTFtreated than in vehicle-treated ischemic gerbils (Fig. 3) (50 rig/day CNTF versus vehicle in the individual strata of ischemic animals: V = 7, P < 0.01; V = 13, P < 0.01; V = 14.5, P c 0.025; 500 ngfday CNTF versus vehicle in
Fig. 3. Effect of CNTF on the number of synapses in three strata of the hippocampal CA1 region. Synapses in the strata molecuhue, lacunosumkadiatum 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 hippocampus was prevented significantly when CNTF (50 &day or 500 rig/day) was infused into the lateral ventricle for 7 days. Each value represents median k interquartile range (n = 8-11). *P < 0.025, **P < 0.01, significantly different from the ischemic group with vehicle infusion. Open column, sham operation; closed columns, ischemia.
the individual strata of ischemic animals: V = 3, P < 0.01; V = 5, P < 0.01; V = 8, P < 0.01). During forebrain ischemia, brain temperature has been shown to fall to different degrees in individual animals, thereby affecting the number of viable CA1 neurons after ischemia [lo]. To avoid the effect of unstable brain temloss, we kept the brain perature on ischemic neuronal temperature at 37.0 f 0.2”C while clamping the common carotid arteries [ 101. This enabled us to apply the same intensity of ischemic insult to all animals and to evaluate with accuracy the neuroprotective effect of CNTF in gerbils with 3-min ischemia. The mechanism(s) by which CNTF exerts a neuroprotective effect on ischemic hippocampal CA1 neurons is not fully understood, although it is possible that CNTF has neurotrophic activity. Brain ischemia has been shown to induce excessive release of endogenous glutamate which further facilitates calcium influx into the cytoplasm of ischemic neurons by binding to N-methyl-D-aspartate receptor [3,12]. Skaper et al. El71 demonstrated that CNTF raises the threshold of hippocampal pyramidal neuron sensitivity to glutamate toxicity, thereby protecting cultured hippocampal neurons against injury due to energy substrate deprivation and excessive glutamate. It is plausible to ascribe the neurotrophic action of CNTF on ischemic CA1 neurons in this study to the suppression by CNTF of neuronal sensitivity to increased extracellular glutamate during and after ischemia. Recently, a functional CNTF receptor (a-receptor) was found in neurons of the hippocampus [7]. This may indicate that CNTF acts on hippocampal neurons by binding to its receptor on the cell surface. In conclusion, the present study first demonstrated that CNTF, when continuously infused into the lateral ventricles of gerbils with 3-min forebrain ischemia, prevents the occurrence of learning disability based on a passive avoidance task and hippocampal neuron loss.
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T.-C. Wen et al. I Neuroscience Letters 19P(1995) 55-58
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 the Uehara Memorial Foundation. [l] 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. [Z] Barbin, G., Manthorpe, M. and Varon, S., Purification of the chick eye ciliary neurotrophic factor, J. Neurochem., 43 (1984) 1468-1478. [3] Choi, D.W. and Rothman, S.M., The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death, Annu. Rev. Neurosci., 13 (1990) 171-182. [4] Clatterbuck, R.E., Price, D.L. and Koliatsos, V.E., Ciliary neurotrophic factor prevents retrograde neuronal death in the adult central nervous system, Proc. Natl. Acad. Sci. USA, 90 (1993) 2222-2226. 151 Hughes, S.M., Lillien, L.E., Raff, M.C., Rohrer, H. and Sendtner, M., Ciliary neurotrophic factor induces type-2 astrocyte differentiation in culture, Nature, 335 (1988) 70-73. 161 Ip, N.Y., Li, Y., van de Stadt, I., Panayotatos, N., Alderson, R.F. and Lindsay, R.M., Ciliary neurotrophic factor enhances neuronal survival in embryonic rat hippocampal cultures, J. Neurosci., 11 (1991) 31243134. [7] Ip, N.Y., McClain, J., Barrezueta, N.X., Aldrich, T.H., Pan, L., Li, Y., Wiegand, S.J., Friedman, B., Davis, S. and Yancopoulos, G.D., The a-component of the CNTF receptor is required for signaling and defines potential CNTF targets in the adult and during development, Neuron, 10 (1993) 89-102. [S] Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 237 (1982) 57-69. [9] Lillien, L.E., Sendtner, M., Rohrer, H., Hughes, SM. and Raff,
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