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Delayed expression of c-fos protein in rat hippocampus and cerebral cortex following transient in vivo exposure to hypoxia
BRAIN RESEARCH ELSEVIER
Brain Research 640 (1994) 119-125
Research Report
Delayed expression of c-los protein in rat hippocampus and cerebral cortex following transient in vivo exposure to hypoxia Takashi Taniguchi a,,, Reiko Fukunaga a, Yasuji Matsuoka a, Kazuhiro Terai b, Ikuo Tooyama b, Hiroshi Kimura b aDepartment of Neurobiology, Kyoto Pharmaceutical University, Yamashina, Kyoto 607, Japan, b Institute of Molecular Neurobiology, Shiga University of Medical Science, Otsu 520-21, Japan (Accepted 9 November 1993)
Abstract
The time course of c-fos protein expression after hypoxia was examined in rat hippocampus and cerebral cortex using an immunohistochemical method. The rats were exposed to in vivo hypoxia for 30 min in a chamber containing 5% O 2 and 95% N 2. Immediately after the treatment, c-fos protein-like immunoreactivity was observed in the granule cell layer of the dentate gyrus. The change was transient, and the density of immunoreactive cells returned quickly to a control level 3 h after the exposure. However, the density of positive cells was again increased 1 day after hypoxia and reached the maximum 7 days after. In the cerebral cortex, on the other hand, no change was detected in the pattern of staining at any time, with an exception on 21 days after hypoxia. At this period, positively stained neurons were significantly increased in both density and intensity throughout the entire extent of the cerebral cortex including the cingulate gyrus. These results clearly indicate that hypoxia induces different patterns of c-fos protein expression among various regions of the brain. The biphasic pattern seen in the dentate gyrus as well as the delayed expression in the cerebral cortex may be related to delayed neuronal damages induced by hypoxia.
Key words: c-Fos; Hypoxia; Hippocampus; Cortex; Rat
1. Introduction
The c-los gene encodes transcription regulatory factors which mediate long-term responses to transsynaptic signals (for reviews, see refs. 10, 18). Although the c-fos protein (Fos) is normally present in the brain, its expression is increased rapidly but transiently in response to several stimuli or brain damages including electrical [16] and sensory or noxious stimulation [2,5,6,22], seizure [3,9,14] and brain ischemia [7,12,19, 20,21]. After brain ischemia, Fos is dramatically upregulated in the CA1 hippocampal neurons [7]. Since those CA1 neurons are highly vulnerable to delayed neuronal death [8,15], the Fos expression has been thought to be a temporal profile for delayed neuronal loss linked to excitotoxic mechanism [7]. This hypothesis is supported by data showing that the Fos expression is enhanced by excitatory amino acids [14] and is
* Corresponding author. Fax: (81) 75-595-4796. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 5 2 8 - B
suppressed by N-methyl-D-aspartate ( N M D A ) receptor antagonists [19]. Therefore, Fos immunoreactivity may provide a sensitive marker for the neuronal vulnerability at the cellular level [7,16]. The aim to use the animal models of brain ischemia is to reproduce hypoxic or anoxic conditions which possibly occur in some brain diseases of human. Most of these models, however, require surgical invasion with unavoidable strong noxious stimuli which would eventually cause high levels of the Fos expression. Thus, the Fos expression in animal models of brain ischemia must be carefully evaluated. Recently we have established an animal model for brain hypoxia [11] which offers reproducible results with no inherent noxious stimuli. Using the hypoxic rat, we have previously shown that hypoxia causes an upregulation of phosphoinositide (PI) turnover which is particularly evident in the hippocampus [11]. Activation of PI turnover is known to lead to an increase in intracellular Ca 2+ and formation of arachidonic acid [1,14]. Both these substances act neurotoxic when accumulated in high con-
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T. Taniguchi et al. / Brain Research 640 (1994) 119-125
centrations. Therefore, the change in neuronal metabolism after hypoxia may share a common mechanism underlying ischemia-induced neuronal injury in the hippocampus, because ischemia is always accompanied by hypoxia. Our empirical impression indicates, however, that brain regions suffered from neuronal damage may vary between hypoxia and ischemia. In the present study, we examined Fos by immunohistochemistry in hypoxic rats, particularly focusing on the expression in the hippocampus and cerebral cortex. The results will be discussed in comparison with those reported in ischemic animal models.
2. Materials and methods 2.1. Exposure to hypoxia and tissue preparations Seven-week-old male Fisher-344 rats (Chares River Co., Japan) weighing approximately 100 g were used. The animals were fasted overnight with free access to water. On the next morning, the rats were exposed to hypoxia in a chamber as described previously [11]. In brief, a gas mixture of 5% O 2 and 95% N 2 was passed through a chamber at a flow rate of 3 liter/min. CO 2 was eliminated by a soda lime CO 2 scrubber. The concentration of 02 in the chamber was monitored continuously throughout the experiment with an oxygen measuring device and the temperature was kept at 22+ I°C. Rats used for the hypoxic treatment were placed for 30 min in the hypoxic chamber, while rats for controls were kept in a similar chamber filled with room air. Empirically, we have observed that exposures longer than 30 rain lead to unacceptable levels of fatality. At serial time periods (0, 3 h, 1, 7, 21, 56, 84 days) following the 30-rain hypoxia, both experimental and control rats were anesthetized with sodium pentobarbital (100 mg/kg i.p.). The animals were perfused through the left cardiac ventricle with 150 ml of 0.01 M phosphate-buffered saline (pH 7.4), followed by 300 ml of a cold fixative consisting of 4% paraformaldehyde, 0.35% glutaraldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.4). After perfusion, the brain was quickly removed and postfixed for 2 days with a cold fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.4). The brain was then transferred to 15% sucrose solution in 0.1 M phosphate buffer (pH 7.4) containing 0.1% sodium azide at 4°C. Sections were cut into 20 ~m-thickness in a cryostat (Yamato, Japan) and collected in 0.1 M phosphate-buffered saline (pH 7.4) containing 0.3% Triton-X 100 (PBST). After washing several times, the sections were stored until use in a free-floating state at 4°C.
2.2. c-fos immunostaining The free floating sections were incubated for 3 days with c-fos antibody (diluted 1 : 10,000, Cambridge Res. Biochem., U.K.) at 4°C, then for 2 h at room temperature with biotinylated anti-sheep IgG (diluted 1 : 1,000, Vector Labs., USA), and for 1 h at room temperature with avidin-biotin-peroxidase complex (diluted 1:4,000, Vector,
USA). All sera were diluted with PBST, and the sections were always rinsed in PBST after each incubation. The peroxidase activity was revealed by 0.02% 3,3'-diaminobenzidine in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.005% HzO 2 and 0.3% nickel ammonium sulfate. The stained sections were mounted on glass slides, dehydrated, cleared and coverslipped with Entellan. Some sections were stained with hematoxylin-eosin (H-E) for the examination of possible histological changes following hypoxia. To verify the specificity of immunostaining, some sections were incubated with a c-fos antibody pre-absorbed with the peptide (Cambridge Res. Biochem., UK) against which the antibody was raised.
2.3. Semi-quantitative analysis Counting the number of Fos-immunoreactive cells was carried out under a light microscope (object lens, × 10; eye lens, × 10) in eight control and thirty-three experimental rats. The latter hypoxic animals consisted of five in each group of 0 h, 3 h, 1 day, 7 days and 21 days after hypoxia, and four in each group of 56 days and 84 days after hypoxia. We analyzed immunostained sections at the level of -2.56 mm from the bregma, according to the atlas of Paxinos and Watson [13]. In the hippocampal formation, the identification of the CA1, CA3 and CA4 hippocampal regions and the dentate gyrus was confirmed in H-E-stained adjacent or nearby sections. Every section from different rats showed a similar feature which included each of the four regions of the hippocampal formation. Positive cells in a square ruler (corresponding 1 mm 2 area of a section) assembled in the eye lens were counted. In the cerebral cortex, a set of two square regions (a total of 2 mm z in a section) were selected in order to cover all cortical layers each from the cingulate and parietal cortex. The density of positive cells was calculated and expressed as the number of cells per unit area of 1 mm 2. Statistical analysis was performed using paired t-test.
3. Results
3.1. Hematoxylin-eosin (H-E) staining Amongst the rats exposed to hypoxia, a mild morphological alteration was observed in the hippocampal formation at 21 days after hypoxia (Fig. 1). In accordance with our previous report [23], both the CA3 and CA4 regions contained many neurons with abnormal shapes (Fig. 1A,B). Their somata containing picnotic nuclei were shrunken and eosinophilic, and shaped spindle or triangle (Fig. 1B-D). A similar, but less severe, change was seen in the granule cell layer of the dentate gyrus (Fig. 1B, arrow). No such a change, however, was recognized in the CA1 region (Fig. 1B). On the other hand, the cerebral cortex appeared free from neuronal damage in every cortical region of all the rats examined at various periods after hypoxia (data not shown).
Fig. 1. Photomicrographs of hematoxylin-eosin (H-E)-stained sections of rat hippocampal formation taken from control (A) and 21 days after hypoxia (B). In B, degenerative neurons are seen in the pyramidal layer of CA4 (box C), CA3 (box D) and in the granule cell layer of the dentate gyrus (arrow). The CA1 region, on the other hand, appears free from such a degenerative change. C and D are large magnification of the respective boxed areas in B. Bar = 500/zm for A and B, 100/~m for C and D.
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121
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0
"1~ 400 C m D I~
0¢- 3 0 -
CA1 CA3 CA4 dentate gyrus
E
E
--200 I 0 ~10.
~0. Z
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Oh
3h
ld
7d
21d
56d
84d
Time altar Hypoxia
Fig. 2. Time course of the densityof Fos-immunoreactivecells in the CA1, CA3, CA4 and dentate gyrus. * P < 0.05, * * P < 0.01, significantly different from corresponding values of the control group (n = 4-7).
3.2. Fos i m m u n o s t a i n i n g
The time courses of the density of Fos-immunopositive cells in the hippocampal formation and the cerebral cortex are shown in Figs. 2 and 4, respectively. Typical examples of positive cells in the two regions are shown in Figs. 3 and 5 with 7 photomicrographs. In control animals, a few positive neurons were distributed sporadically in both hippocampus formation and cerebral cortex (Figs. 3A and 5A). In these positive neurons, reaction products were localized to
their nuclear entities (Figs. 3A and 5A). This result in intact rats agrees well with previous reports [3,8,16]. In hypoxia-treated rats, the Fos expression was increased in many forebrain regions including various thalamic, hypothalamic and amygdaloid nuclei. The detail of the expression pattern in these structures will be described elsewhere. In the present study, we analyzed the Fos expression morphometrically in the hippocampus and cerebral cortex. The response of the Fos expression differed markedly among different regions of the hippocampal formation. The Fos expression was increased only in the granule cell layer of the dentate gyrus but not in the hippocampus including the CA1, CA3 and CA4 regions (Fig. 2). The density of positive cells in the granule cell layer, as compared with that in the control (mean + S.E.; 10.5 + 2.5 cells/mm2), was elevated significantly at 0 h after hypoxia (32.8 + 5.6; Figs. 2 and 3B) but returned soon to the control level 3 h after the treatment (13.2 + 1.6; Figs. 2 and 3C). Interestingly enough, however, the cell density was again increased significantly after 1 day (24.0 + 6.9) and reached the maximum after 7 days (31.2 + 6.5; Figs. 2 and 3D). In the cerebral cortex, on the other hand, there was no increase in the density of positive cells by 7 days after hypoxia (Figs. 4 and 5). On day 21, however, a transient increase of positive cell density was observed throughout the entire rostrocaudal extent of the cerebral cortex including the neocortical parietal cortex (4.17-fold the control value in means; Figs. 4 and 5C) and mesocortical cingulate gyrus (3.99-folds of
Fig. 3. Photomicrographsof Fos-immunostainingin the dentate gyrusfrom a control rat (A), and from hypoxicrats examined0 h (B), 3 h (C) and 7 days (D) after hypoxia. Bar = 200/xm.
T. Taniguchi et al. /Brain Research 640 (1994) 119-125 ffl (D
140-
o "ID 120C
. n
1000 80-
E
r-hA
.EE 60¢) 0 U. 40I 0 '~ 2 0 -
E Z
0-
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7d 21d 56d 84d
Time after Hypoxia
Fig. 4. Time course of the density of Fos-immunoreactive cells in the cerebral cortex. The cingulate cortex indicated by A and the parietal cortex indicated by B are analyzed. * * P < 0.01, significantly different from corresponding values of the control group (n = 4-6).
the control value in means; Fig. 4). Similarly to these data shown in Fig. 5C, many positive cells were irregularly distributed from shallow to deep layers of other neocortical regions (data not shown).
4. Discussion
It has been known that neurons in the CA1 region are damaged by ischemia, while almost all neurons in the dentate gyrus, CA3 and CA4 are preserved [8]. However, as reported previously [23] and also here, the examination with H-E staining has proved that hypoxia induces degenerative changes (shrunken and eosinophilic somata with picnotic nuclei) in neurons not of the CA1 but of the dentate gyrus, CA3 and CA4 on 21 days after the treatment. This clearly indicates that brain regions sensitive to hypoxia are different from those vulnerable to the treatment in animal models of ischemia. The present study demonstrates that hypoxia enhances the Fos expression, with a biphasic time course in the dentate gyrus whereas with a tardy and transient course in the cerebral cortex. Other forebrain regions revealing the Fos expression following hypoxia include various hypothalamic and thalamic nuclei, amygdala, but not piriform cortex (data not shown). It may be worthy to emphasize that there appears to be no correlation between the regions in which Fos is induced with hypoxia and the regions in which evidence of neuronal damage occurs as judged histologically. The pattern of the Fos expression appears somehow different from that after seizures [3,9] or ischemia [7,12,19,20,21]. For example, Dragunow et al. [3] reported that kindling stimulation induced c-fos protein not only in the den-
123
tate gyrus but also in the piriform cortex, CA1, CA3 and CA4 regions. Uemura et al. [19] reported that the Fos expression was observed mainly in the dentate gyrus, CA3 and CA4, and partly in the CA1 region after ischemia produced by carotid clamping. These results suggest that different mechanisms for neuronal damage may occur between hypoxia and ischemia or seizures. Further studies will be needed to clarify this issue. In the present study, the initial peak of the Fos expression in the dentate gyrus is seen at 0 h after hypoxia. Previous studies have shown that the Fos expression is detectable 10-15 min after neuronal damage [10,19]. It is therefore likely that, in our experiment, neuronal damage may have started during the 30 min when the rats were staying in the chamber. After the initial peak, the Fos expression in the dentate gyrus returns to the control level within 3 h. This time course is similar to that seen in the seizure induced by metrazole [9]. It is of great interest, however, that the hypoxia-induced Fos expression is again increased after 1 day. Such an up-regulation in the dentate gyrus continues for at least several days. At present, the significance of this biphasic Fos expression remains unclear. According to a previous study [11], however, the PI turnover in the dentate gyrus is kept at this enhanced state following hypoxia for over 24 h. In addition, the up-regulation of PI is known to lead to an increase in intracellular Ca 2÷ influx [1,4]. It is thus suggested that the delayed second peak found here in the dentate gyrus is associated with post-hypoxic hyperexcitability which may cause delayed neuronal injury. In the cerebral cortex, the Fos expression in this study is seen in the entire extent of the neocortex and mesocortical cingulate gyrus. Such a wide distribution of Fos expression in the cortex may reflect an extensive influence of in vivo hypoxia employed. Since the piriform and entorhinal cortex which are known to express high levels of Fos following epileptic seizures are kept unchanged, these cortices appear insensitive to hypoxia. The neo- and mesocortical Fos expressions are significantly increased only on day 21 after hypoxia. Previous studies have also shown that the Fos expression in the cortex is slightly delayed as compared with that in the dentate gyrus [3,9]. However, the time course after hypoxia is much slower. The reason for the very long delay in the cortex remains unclear. It may be worthy to note that, on day 21 after hypoxia, degenerative neurons are recognizable in the CA3 and CA4 regions of H-E-stained preparations. Therefore, delayed neuronal damage, though not detectable with the technique employed, may occur more or less in some cortical neurons. Another view is that the enhanced Fos expression occurs in the dentate gyrus or the cerebral cortex which possess slight or no neuronal damage, respectively, while Fos expression is minimally
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T. Taniguchi et al. /Brain Research 640 (1994) 119-125
Fig. 5. Photomicrographsof Fos-immunostainingin the cingulate cortex from a control rat (A), and from hypoxicrats examined 0 hour (B) and 21 days (C) after hypoxia. Bar = 200/~m.
increased in the CA3 and CA4 where severe neuronal degeneration has been brought about. A possible interpretation from this point of view is that c-fos expression may be a protective event in these brain regions. Currently, it is unclear whether the same gene is being expressed at short and long periods of time after hypoxic gas exposure. It is possible that the anitoby used in this study recognize not only Fos but also Fos-related antigens or some other members of Fos-
protein family. Sharp et al. [17] showed that osmotic stimuli induced c-los acutely and then induced Fos-related antigens after some delay. The question whether the staining represented Fos remains to be elucidated in future study using biochemical Northern blots or in situ hybridization histochemistry. Finally, it should be pointed out that the animal model used here involves the aspiration of CO 2 free hypoxic gas. The effect of such a gas must be supplied
T. Taniguchi et al. /Brain Research 640 (1994) 119-125
not only to the brain but also to various peripheral organs such as the liver. It is possible, therefore, that some compounds released from these organs by the aspiration may have potentially affected brain neurons secondarily. It is also possible that the elimination of CO 2 in the hypoxic gas has affected brain cells in a manner different from that occurring in ischemic animal models. Although CO2 gas in different concentrations may contribute differently to neuronal injury, the present animal model should easily permit a future analysis with various compositions of a hypoxic gas. Acknowledgments. This paper was partially supported by Grant-inAid for Scientific Research by Ministry of Education, Science and Culture, Japan (T.T. and H.K.), Uehara Medical Foundation (I.T.), Sasakawa Medical Research Foundation (I.T.) and Shiga Medical Science Association for International Corporation (I.T.).
5. References [1] Abdel-Latif, A.A., Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers, Pharmacol. Rev., 38 (1986) 227-272. [2] BuUitt, E., Induction of c-fos-like protein within the lumbar spinal cord and thalamus of the rat following peripheral stimulation, Brain Res., 493 (1989) 391-397. [3] Dragunow, M. and Robertoson, H.A., Kindling stimulation induces c-los protein(s) in granule cells of the rat dentate gyrus, Nature, 329 (1987) 441-442. [4] Fisher, S.K. and Agranoff, B.W., Receptor activation and inositol lipid hydrolysis in neural tissue, J. Neurochem., 48 (1987) 999-1017. [5] Herdegen, T., Kovary, K., Leah, J. and Bravo, R., Specific temporal and spatial distribution of Jun, Fos, and Krox-24 proteins in spinal neurons following noxious transsynaptic stimulation, Z Comp. Neurol., 313 (1991) 178-191. [6] Hunt, S.P., Pini, A. and Evan, G., Induction of c-fos-like protein in spinal cord neurons following sensory stimulation, Nature, 328 (1987) 632-634. [7] Jorgensen, M.B., Deckert, J., Wright, D.C. and Gehlert, D.R., Delayed c-fos proto-oncogene expression in the rat hippocampus induced by transient global cerebral ischemia: an in situ hybridization study, Brain Res., 484 (1989) 393-398. [8] Kirino, T., Delayed neuronal death in the gerbile hippocampus following ischemia, Brain Res., 239 (1982) 57-69.
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[9] Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Currari, T., Mapping patterns of c-fos expression in the central nervous system after seizure, Science, 237 (1987) 192-196. [10] Morgan, J.l. and Curran, T., Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Trends Neurosci., 12 (1989) 459-462. [11] Ninomiya, H., Taniguchi, T., Fujiwara, M., Shimohama, S. and Kameyama, M., Effect of in vivo exposure to hypoxia on muscarinic cholinergic receptor-coupled phosphoinositide turnover in the rat brain, Brain Res., 482 (1989) 109-121. [12] Onodera, H., Kogure, K., Ono, Y., Igarashi, K., Kirino, Y. and Nagaoka, A., Proto-oncogene c-fos is transiently induced in the rat cerebral cortex after forebrain ischemia, Neurosci. Letb, 98 (1989) 101-104. [13] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1982. [14] Popovici, T., Barbin, G. and Ben-Ari, Y., Kainic acid induced seizures increase c-fos-like protein in rat hippocampus. Eur. J. Pharmacol., 150 (1988) 405-406. [15] Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. NeuroL, 11 (1982) 491-498. [16] Sagar, S.M., Sharp, F.R. and Curran, T., Expression of c-fos protein in brain: Metabolic mapping at the cellular level. Science, 240 (1988) 1328-1331. [17] Sharp, F.R., Sagar S.M., Hicks, K., Lowenstein D. and Hisanaga K., c-fos mRNA, Fos, and Fos-related antigen induction by hypertonic saline and stress, J. Neurosci., 11 (1991) 2321-2331. [18] Sheng, M. and Greenberg, M.E., The regulation and function of c-fos and other immediate early genes in the nervous system, Neuron, 4 (1990) 477-485. [19] Uemura, Y., Kowall, N.W. and Flint Beal, M., Global ischemia induces NMDA receptor-mediated c-fos expression in neurons resistant injury in gerbil hippocampus, Brain Res., 542 (1991) 343-347. [20] Uemura, Y., Kowall, N.W. and Moskouitz, M.A., Focal ischemia in rats causes time-dependent expression of c-fos protein immunoreactivity in wide spread regions of ipsilateral cortex. Brain Res., 552 (1991) 99-105. [21] Wessel, T.C., Joh, T.H. and Volpe, B.T., In situ hybridization analysis of c-fos and c-jun expression in the rat brain following transient forebrain ischemia, Brain Res., 567 (1991) 231-240. [22] Williams, S., Evan, G.I. and Hunt, S.P., Changing patterns of c-fos induction in spinal neurons following thermal cutaneous stimulation in the rat, Neuroscience, 36 (1990) 73-81. [23] Yamaoka, Y., Shimohama, S., Kimura, J., Fukunaga, R. and Taniguchi, T., Neuronal damage in the rat hippocampus induced by in vivo hypoxia, Exp. Toxicol. Pathol., 45 (1993) 205209.
Brain Research 640 (1994) 119-125
Research Report
Delayed expression of c-los protein in rat hippocampus and cerebral cortex following transient in vivo exposure to hypoxia Takashi Taniguchi a,,, Reiko Fukunaga a, Yasuji Matsuoka a, Kazuhiro Terai b, Ikuo Tooyama b, Hiroshi Kimura b aDepartment of Neurobiology, Kyoto Pharmaceutical University, Yamashina, Kyoto 607, Japan, b Institute of Molecular Neurobiology, Shiga University of Medical Science, Otsu 520-21, Japan (Accepted 9 November 1993)
Abstract
The time course of c-fos protein expression after hypoxia was examined in rat hippocampus and cerebral cortex using an immunohistochemical method. The rats were exposed to in vivo hypoxia for 30 min in a chamber containing 5% O 2 and 95% N 2. Immediately after the treatment, c-fos protein-like immunoreactivity was observed in the granule cell layer of the dentate gyrus. The change was transient, and the density of immunoreactive cells returned quickly to a control level 3 h after the exposure. However, the density of positive cells was again increased 1 day after hypoxia and reached the maximum 7 days after. In the cerebral cortex, on the other hand, no change was detected in the pattern of staining at any time, with an exception on 21 days after hypoxia. At this period, positively stained neurons were significantly increased in both density and intensity throughout the entire extent of the cerebral cortex including the cingulate gyrus. These results clearly indicate that hypoxia induces different patterns of c-fos protein expression among various regions of the brain. The biphasic pattern seen in the dentate gyrus as well as the delayed expression in the cerebral cortex may be related to delayed neuronal damages induced by hypoxia.
Key words: c-Fos; Hypoxia; Hippocampus; Cortex; Rat
1. Introduction
The c-los gene encodes transcription regulatory factors which mediate long-term responses to transsynaptic signals (for reviews, see refs. 10, 18). Although the c-fos protein (Fos) is normally present in the brain, its expression is increased rapidly but transiently in response to several stimuli or brain damages including electrical [16] and sensory or noxious stimulation [2,5,6,22], seizure [3,9,14] and brain ischemia [7,12,19, 20,21]. After brain ischemia, Fos is dramatically upregulated in the CA1 hippocampal neurons [7]. Since those CA1 neurons are highly vulnerable to delayed neuronal death [8,15], the Fos expression has been thought to be a temporal profile for delayed neuronal loss linked to excitotoxic mechanism [7]. This hypothesis is supported by data showing that the Fos expression is enhanced by excitatory amino acids [14] and is
* Corresponding author. Fax: (81) 75-595-4796. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved
SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 5 2 8 - B
suppressed by N-methyl-D-aspartate ( N M D A ) receptor antagonists [19]. Therefore, Fos immunoreactivity may provide a sensitive marker for the neuronal vulnerability at the cellular level [7,16]. The aim to use the animal models of brain ischemia is to reproduce hypoxic or anoxic conditions which possibly occur in some brain diseases of human. Most of these models, however, require surgical invasion with unavoidable strong noxious stimuli which would eventually cause high levels of the Fos expression. Thus, the Fos expression in animal models of brain ischemia must be carefully evaluated. Recently we have established an animal model for brain hypoxia [11] which offers reproducible results with no inherent noxious stimuli. Using the hypoxic rat, we have previously shown that hypoxia causes an upregulation of phosphoinositide (PI) turnover which is particularly evident in the hippocampus [11]. Activation of PI turnover is known to lead to an increase in intracellular Ca 2+ and formation of arachidonic acid [1,14]. Both these substances act neurotoxic when accumulated in high con-
120
T. Taniguchi et al. / Brain Research 640 (1994) 119-125
centrations. Therefore, the change in neuronal metabolism after hypoxia may share a common mechanism underlying ischemia-induced neuronal injury in the hippocampus, because ischemia is always accompanied by hypoxia. Our empirical impression indicates, however, that brain regions suffered from neuronal damage may vary between hypoxia and ischemia. In the present study, we examined Fos by immunohistochemistry in hypoxic rats, particularly focusing on the expression in the hippocampus and cerebral cortex. The results will be discussed in comparison with those reported in ischemic animal models.
2. Materials and methods 2.1. Exposure to hypoxia and tissue preparations Seven-week-old male Fisher-344 rats (Chares River Co., Japan) weighing approximately 100 g were used. The animals were fasted overnight with free access to water. On the next morning, the rats were exposed to hypoxia in a chamber as described previously [11]. In brief, a gas mixture of 5% O 2 and 95% N 2 was passed through a chamber at a flow rate of 3 liter/min. CO 2 was eliminated by a soda lime CO 2 scrubber. The concentration of 02 in the chamber was monitored continuously throughout the experiment with an oxygen measuring device and the temperature was kept at 22+ I°C. Rats used for the hypoxic treatment were placed for 30 min in the hypoxic chamber, while rats for controls were kept in a similar chamber filled with room air. Empirically, we have observed that exposures longer than 30 rain lead to unacceptable levels of fatality. At serial time periods (0, 3 h, 1, 7, 21, 56, 84 days) following the 30-rain hypoxia, both experimental and control rats were anesthetized with sodium pentobarbital (100 mg/kg i.p.). The animals were perfused through the left cardiac ventricle with 150 ml of 0.01 M phosphate-buffered saline (pH 7.4), followed by 300 ml of a cold fixative consisting of 4% paraformaldehyde, 0.35% glutaraldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.4). After perfusion, the brain was quickly removed and postfixed for 2 days with a cold fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate buffer (pH 7.4). The brain was then transferred to 15% sucrose solution in 0.1 M phosphate buffer (pH 7.4) containing 0.1% sodium azide at 4°C. Sections were cut into 20 ~m-thickness in a cryostat (Yamato, Japan) and collected in 0.1 M phosphate-buffered saline (pH 7.4) containing 0.3% Triton-X 100 (PBST). After washing several times, the sections were stored until use in a free-floating state at 4°C.
2.2. c-fos immunostaining The free floating sections were incubated for 3 days with c-fos antibody (diluted 1 : 10,000, Cambridge Res. Biochem., U.K.) at 4°C, then for 2 h at room temperature with biotinylated anti-sheep IgG (diluted 1 : 1,000, Vector Labs., USA), and for 1 h at room temperature with avidin-biotin-peroxidase complex (diluted 1:4,000, Vector,
USA). All sera were diluted with PBST, and the sections were always rinsed in PBST after each incubation. The peroxidase activity was revealed by 0.02% 3,3'-diaminobenzidine in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.005% HzO 2 and 0.3% nickel ammonium sulfate. The stained sections were mounted on glass slides, dehydrated, cleared and coverslipped with Entellan. Some sections were stained with hematoxylin-eosin (H-E) for the examination of possible histological changes following hypoxia. To verify the specificity of immunostaining, some sections were incubated with a c-fos antibody pre-absorbed with the peptide (Cambridge Res. Biochem., UK) against which the antibody was raised.
2.3. Semi-quantitative analysis Counting the number of Fos-immunoreactive cells was carried out under a light microscope (object lens, × 10; eye lens, × 10) in eight control and thirty-three experimental rats. The latter hypoxic animals consisted of five in each group of 0 h, 3 h, 1 day, 7 days and 21 days after hypoxia, and four in each group of 56 days and 84 days after hypoxia. We analyzed immunostained sections at the level of -2.56 mm from the bregma, according to the atlas of Paxinos and Watson [13]. In the hippocampal formation, the identification of the CA1, CA3 and CA4 hippocampal regions and the dentate gyrus was confirmed in H-E-stained adjacent or nearby sections. Every section from different rats showed a similar feature which included each of the four regions of the hippocampal formation. Positive cells in a square ruler (corresponding 1 mm 2 area of a section) assembled in the eye lens were counted. In the cerebral cortex, a set of two square regions (a total of 2 mm z in a section) were selected in order to cover all cortical layers each from the cingulate and parietal cortex. The density of positive cells was calculated and expressed as the number of cells per unit area of 1 mm 2. Statistical analysis was performed using paired t-test.
3. Results
3.1. Hematoxylin-eosin (H-E) staining Amongst the rats exposed to hypoxia, a mild morphological alteration was observed in the hippocampal formation at 21 days after hypoxia (Fig. 1). In accordance with our previous report [23], both the CA3 and CA4 regions contained many neurons with abnormal shapes (Fig. 1A,B). Their somata containing picnotic nuclei were shrunken and eosinophilic, and shaped spindle or triangle (Fig. 1B-D). A similar, but less severe, change was seen in the granule cell layer of the dentate gyrus (Fig. 1B, arrow). No such a change, however, was recognized in the CA1 region (Fig. 1B). On the other hand, the cerebral cortex appeared free from neuronal damage in every cortical region of all the rats examined at various periods after hypoxia (data not shown).
Fig. 1. Photomicrographs of hematoxylin-eosin (H-E)-stained sections of rat hippocampal formation taken from control (A) and 21 days after hypoxia (B). In B, degenerative neurons are seen in the pyramidal layer of CA4 (box C), CA3 (box D) and in the granule cell layer of the dentate gyrus (arrow). The CA1 region, on the other hand, appears free from such a degenerative change. C and D are large magnification of the respective boxed areas in B. Bar = 500/zm for A and B, 100/~m for C and D.
T. Taniguchi et al. /Brain Research 640 (1994) 119-125
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0
"1~ 400 C m D I~
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CA1 CA3 CA4 dentate gyrus
E
E
--200 I 0 ~10.
~0. Z
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Oh
3h
ld
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21d
56d
84d
Time altar Hypoxia
Fig. 2. Time course of the densityof Fos-immunoreactivecells in the CA1, CA3, CA4 and dentate gyrus. * P < 0.05, * * P < 0.01, significantly different from corresponding values of the control group (n = 4-7).
3.2. Fos i m m u n o s t a i n i n g
The time courses of the density of Fos-immunopositive cells in the hippocampal formation and the cerebral cortex are shown in Figs. 2 and 4, respectively. Typical examples of positive cells in the two regions are shown in Figs. 3 and 5 with 7 photomicrographs. In control animals, a few positive neurons were distributed sporadically in both hippocampus formation and cerebral cortex (Figs. 3A and 5A). In these positive neurons, reaction products were localized to
their nuclear entities (Figs. 3A and 5A). This result in intact rats agrees well with previous reports [3,8,16]. In hypoxia-treated rats, the Fos expression was increased in many forebrain regions including various thalamic, hypothalamic and amygdaloid nuclei. The detail of the expression pattern in these structures will be described elsewhere. In the present study, we analyzed the Fos expression morphometrically in the hippocampus and cerebral cortex. The response of the Fos expression differed markedly among different regions of the hippocampal formation. The Fos expression was increased only in the granule cell layer of the dentate gyrus but not in the hippocampus including the CA1, CA3 and CA4 regions (Fig. 2). The density of positive cells in the granule cell layer, as compared with that in the control (mean + S.E.; 10.5 + 2.5 cells/mm2), was elevated significantly at 0 h after hypoxia (32.8 + 5.6; Figs. 2 and 3B) but returned soon to the control level 3 h after the treatment (13.2 + 1.6; Figs. 2 and 3C). Interestingly enough, however, the cell density was again increased significantly after 1 day (24.0 + 6.9) and reached the maximum after 7 days (31.2 + 6.5; Figs. 2 and 3D). In the cerebral cortex, on the other hand, there was no increase in the density of positive cells by 7 days after hypoxia (Figs. 4 and 5). On day 21, however, a transient increase of positive cell density was observed throughout the entire rostrocaudal extent of the cerebral cortex including the neocortical parietal cortex (4.17-fold the control value in means; Figs. 4 and 5C) and mesocortical cingulate gyrus (3.99-folds of
Fig. 3. Photomicrographsof Fos-immunostainingin the dentate gyrusfrom a control rat (A), and from hypoxicrats examined0 h (B), 3 h (C) and 7 days (D) after hypoxia. Bar = 200/xm.
T. Taniguchi et al. /Brain Research 640 (1994) 119-125 ffl (D
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. n
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E
r-hA
.EE 60¢) 0 U. 40I 0 '~ 2 0 -
E Z
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Fig. 4. Time course of the density of Fos-immunoreactive cells in the cerebral cortex. The cingulate cortex indicated by A and the parietal cortex indicated by B are analyzed. * * P < 0.01, significantly different from corresponding values of the control group (n = 4-6).
the control value in means; Fig. 4). Similarly to these data shown in Fig. 5C, many positive cells were irregularly distributed from shallow to deep layers of other neocortical regions (data not shown).
4. Discussion
It has been known that neurons in the CA1 region are damaged by ischemia, while almost all neurons in the dentate gyrus, CA3 and CA4 are preserved [8]. However, as reported previously [23] and also here, the examination with H-E staining has proved that hypoxia induces degenerative changes (shrunken and eosinophilic somata with picnotic nuclei) in neurons not of the CA1 but of the dentate gyrus, CA3 and CA4 on 21 days after the treatment. This clearly indicates that brain regions sensitive to hypoxia are different from those vulnerable to the treatment in animal models of ischemia. The present study demonstrates that hypoxia enhances the Fos expression, with a biphasic time course in the dentate gyrus whereas with a tardy and transient course in the cerebral cortex. Other forebrain regions revealing the Fos expression following hypoxia include various hypothalamic and thalamic nuclei, amygdala, but not piriform cortex (data not shown). It may be worthy to emphasize that there appears to be no correlation between the regions in which Fos is induced with hypoxia and the regions in which evidence of neuronal damage occurs as judged histologically. The pattern of the Fos expression appears somehow different from that after seizures [3,9] or ischemia [7,12,19,20,21]. For example, Dragunow et al. [3] reported that kindling stimulation induced c-fos protein not only in the den-
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tate gyrus but also in the piriform cortex, CA1, CA3 and CA4 regions. Uemura et al. [19] reported that the Fos expression was observed mainly in the dentate gyrus, CA3 and CA4, and partly in the CA1 region after ischemia produced by carotid clamping. These results suggest that different mechanisms for neuronal damage may occur between hypoxia and ischemia or seizures. Further studies will be needed to clarify this issue. In the present study, the initial peak of the Fos expression in the dentate gyrus is seen at 0 h after hypoxia. Previous studies have shown that the Fos expression is detectable 10-15 min after neuronal damage [10,19]. It is therefore likely that, in our experiment, neuronal damage may have started during the 30 min when the rats were staying in the chamber. After the initial peak, the Fos expression in the dentate gyrus returns to the control level within 3 h. This time course is similar to that seen in the seizure induced by metrazole [9]. It is of great interest, however, that the hypoxia-induced Fos expression is again increased after 1 day. Such an up-regulation in the dentate gyrus continues for at least several days. At present, the significance of this biphasic Fos expression remains unclear. According to a previous study [11], however, the PI turnover in the dentate gyrus is kept at this enhanced state following hypoxia for over 24 h. In addition, the up-regulation of PI is known to lead to an increase in intracellular Ca 2÷ influx [1,4]. It is thus suggested that the delayed second peak found here in the dentate gyrus is associated with post-hypoxic hyperexcitability which may cause delayed neuronal injury. In the cerebral cortex, the Fos expression in this study is seen in the entire extent of the neocortex and mesocortical cingulate gyrus. Such a wide distribution of Fos expression in the cortex may reflect an extensive influence of in vivo hypoxia employed. Since the piriform and entorhinal cortex which are known to express high levels of Fos following epileptic seizures are kept unchanged, these cortices appear insensitive to hypoxia. The neo- and mesocortical Fos expressions are significantly increased only on day 21 after hypoxia. Previous studies have also shown that the Fos expression in the cortex is slightly delayed as compared with that in the dentate gyrus [3,9]. However, the time course after hypoxia is much slower. The reason for the very long delay in the cortex remains unclear. It may be worthy to note that, on day 21 after hypoxia, degenerative neurons are recognizable in the CA3 and CA4 regions of H-E-stained preparations. Therefore, delayed neuronal damage, though not detectable with the technique employed, may occur more or less in some cortical neurons. Another view is that the enhanced Fos expression occurs in the dentate gyrus or the cerebral cortex which possess slight or no neuronal damage, respectively, while Fos expression is minimally
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Fig. 5. Photomicrographsof Fos-immunostainingin the cingulate cortex from a control rat (A), and from hypoxicrats examined 0 hour (B) and 21 days (C) after hypoxia. Bar = 200/~m.
increased in the CA3 and CA4 where severe neuronal degeneration has been brought about. A possible interpretation from this point of view is that c-fos expression may be a protective event in these brain regions. Currently, it is unclear whether the same gene is being expressed at short and long periods of time after hypoxic gas exposure. It is possible that the anitoby used in this study recognize not only Fos but also Fos-related antigens or some other members of Fos-
protein family. Sharp et al. [17] showed that osmotic stimuli induced c-los acutely and then induced Fos-related antigens after some delay. The question whether the staining represented Fos remains to be elucidated in future study using biochemical Northern blots or in situ hybridization histochemistry. Finally, it should be pointed out that the animal model used here involves the aspiration of CO 2 free hypoxic gas. The effect of such a gas must be supplied
T. Taniguchi et al. /Brain Research 640 (1994) 119-125
not only to the brain but also to various peripheral organs such as the liver. It is possible, therefore, that some compounds released from these organs by the aspiration may have potentially affected brain neurons secondarily. It is also possible that the elimination of CO 2 in the hypoxic gas has affected brain cells in a manner different from that occurring in ischemic animal models. Although CO2 gas in different concentrations may contribute differently to neuronal injury, the present animal model should easily permit a future analysis with various compositions of a hypoxic gas. Acknowledgments. This paper was partially supported by Grant-inAid for Scientific Research by Ministry of Education, Science and Culture, Japan (T.T. and H.K.), Uehara Medical Foundation (I.T.), Sasakawa Medical Research Foundation (I.T.) and Shiga Medical Science Association for International Corporation (I.T.).
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[9] Morgan, J.I., Cohen, D.R., Hempstead, J.L. and Currari, T., Mapping patterns of c-fos expression in the central nervous system after seizure, Science, 237 (1987) 192-196. [10] Morgan, J.l. and Curran, T., Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Trends Neurosci., 12 (1989) 459-462. [11] Ninomiya, H., Taniguchi, T., Fujiwara, M., Shimohama, S. and Kameyama, M., Effect of in vivo exposure to hypoxia on muscarinic cholinergic receptor-coupled phosphoinositide turnover in the rat brain, Brain Res., 482 (1989) 109-121. [12] Onodera, H., Kogure, K., Ono, Y., Igarashi, K., Kirino, Y. and Nagaoka, A., Proto-oncogene c-fos is transiently induced in the rat cerebral cortex after forebrain ischemia, Neurosci. Letb, 98 (1989) 101-104. [13] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1982. [14] Popovici, T., Barbin, G. and Ben-Ari, Y., Kainic acid induced seizures increase c-fos-like protein in rat hippocampus. Eur. J. Pharmacol., 150 (1988) 405-406. [15] Pulsinelli, W.A., Brierley, J.B. and Plum, F., Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann. NeuroL, 11 (1982) 491-498. [16] Sagar, S.M., Sharp, F.R. and Curran, T., Expression of c-fos protein in brain: Metabolic mapping at the cellular level. Science, 240 (1988) 1328-1331. [17] Sharp, F.R., Sagar S.M., Hicks, K., Lowenstein D. and Hisanaga K., c-fos mRNA, Fos, and Fos-related antigen induction by hypertonic saline and stress, J. Neurosci., 11 (1991) 2321-2331. [18] Sheng, M. and Greenberg, M.E., The regulation and function of c-fos and other immediate early genes in the nervous system, Neuron, 4 (1990) 477-485. [19] Uemura, Y., Kowall, N.W. and Flint Beal, M., Global ischemia induces NMDA receptor-mediated c-fos expression in neurons resistant injury in gerbil hippocampus, Brain Res., 542 (1991) 343-347. [20] Uemura, Y., Kowall, N.W. and Moskouitz, M.A., Focal ischemia in rats causes time-dependent expression of c-fos protein immunoreactivity in wide spread regions of ipsilateral cortex. Brain Res., 552 (1991) 99-105. [21] Wessel, T.C., Joh, T.H. and Volpe, B.T., In situ hybridization analysis of c-fos and c-jun expression in the rat brain following transient forebrain ischemia, Brain Res., 567 (1991) 231-240. [22] Williams, S., Evan, G.I. and Hunt, S.P., Changing patterns of c-fos induction in spinal neurons following thermal cutaneous stimulation in the rat, Neuroscience, 36 (1990) 73-81. [23] Yamaoka, Y., Shimohama, S., Kimura, J., Fukunaga, R. and Taniguchi, T., Neuronal damage in the rat hippocampus induced by in vivo hypoxia, Exp. Toxicol. Pathol., 45 (1993) 205209.