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Increase of fragmented DNA transport in apical dendrites of gerbil CA1 pyramidal neurons following transient forebrain ischemia by mild hypothermia
Neuroscience Letters 280 (2000) 73±77 www.elsevier.com/locate/neulet
Increase of fragmented DNA transport in apical dendrites of gerbil CA1 pyramidal neurons following transient forebrain ischemia by mild hypothermia Akira Hara a,*, Masayuki Niwa b, Tomohiko Iwai c, Hirohito Yano c, Yasuo Bunai d, Toshihiko Uematsu b, Naoki Yoshimi a, Hideki Mori a a Department of Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan Department of Pharmacology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan c Department of Neurosurgery, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan d Department of Legal Medicine, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan b
Received 15 October 1999; received in revised form 2 December 1999; accepted 6 December 1999
Abstract Mild hypothermia (388C) accelerated transport of fragmented DNA in apical dendrites of the gerbil CA1 pyramidal neurons and increased dendrite-terminal fragmented DNA pooling in the apoptotic process following transient forebrain ischemia. The speci®c DNA fragmentation after the ischemic insult in gerbil hippocampus was examined by in situ nickend-labeling method, and ¯uorescence DNA detection technique by DAPI was also performed. There is a precise temperature dependence for the migration of fragmented DNA from nuclei into apical dendrites of CA1 pyramidal cells during apoptosis following transient forebrain ischemia. Increase of fragmented DNA pooling is highly temperature sensitive, occurring at 388C, while at 398C there is a marked decrease in DNA pooling. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Delayed neuronal death; Gerbil; DNA fragmentation; Apical dendrite; Hypothermia
Recently, many studies support the hypothesis that delayed neuronal death (DND) of the CA1 pyramidal neurons after transient forebrain ischemia is not necrotic but apoptotic. Morphological and biochemical data such as DNA ¯uorescence assay, in situ nick-end-labeling method, gel electrophoresis and electron microscope indicate that DND is a form of programmed cell death, or apoptosis. Among them, the most characteristic phenomenon for apoptosis is internucleosomal DNA cleavage into oligonucleosome-length fragments that can be directly visualized by end-labeling with biotinylated dUTP in a reaction that employs terminal deoxynucleotide transferase (TUNEL) [18,19]. In a previous study [10], we histologically demonstrated the migration of the fragmented DNA from the nuclei into the apical dendrite of the CA1 pyramidal neurons of gerbil hippocampus following ischemia, and showed an active transport mechanism of fragmented DNA in the late phase * Corresponding author. Tel.: 181-58-267-2235; fax: 181-58265-9005. E-mail address: [email protected] (A. Hara)
of apoptotic process induced by transient forebrain ischemia. The study focused on not only detection of the nuclear positive reaction of TUNEL, but also on the transportation of fragmented DNA in neuronal ®bers. The phenomenon of the fragmented DNA movement into neuronal ®bers is an evidence of intracellular metabolic mechanisms that occur during the apoptotic process. Hypothermia during transient forebrain ischemia exacerbates ischemia-induced neuronal death in hippocampal CA1 neurons [4,11,15]. Production of reactive oxygen species [15], hydroxyl free radicals [8], calcium in¯ux into neurons [2] and vascular permeability [5] have been reported to be enhanced by hypothermia in neurons of hippocampal CA1 neurons. It is also well known that axoplasmic transportation is very sensitive to temperature conditions [16]. Average transport velocity is exponentially related to temperature over a wide range, although most studies of axoplasmic transport have been performed by using peripheral nerves. There are no reports evaluating the effects of hypothermia on the axoplasmic transportation within the central nervous system.
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 97 9- 9
74
A. Hara et al. / Neuroscience Letters 280 (2000) 73±77
In the present study, using the transient forebrain ischemia model for apoptosis, we evaluated in gerbil CA1 pyramidal neurons the effects of temperatures (37±398C) on fragmented DNA movement (in neuronal ®bers) and pooling (in dendrite terminals). Male Mongolian gerbils, weighing 65±75 g, were subjected to severe forebrain ischemia as described previously [11±13]. Anesthesia was induced with 4% halothane and maintained with 2% halothane during subsequent surgery. Carotid arteries were isolated through an anterior midcervical incision, and animals were subjected to 5 min forebrain ischemia with microclips applied to the carotid arteries. Rectal temperatures of 37, 37.5, 38 or 398C, maintained using a heating pad with a circulating system, were adjusted just before, during and just after ischemic insult. After reperfusion, the temperature was maintained at 378C for 3 h. The rectal temperature was monitored and recorded continuously. If the recording showed that body temperature was not maintained at the pre-de®ned temperature, the animal was excluded from the study. Accordingly, groups of animals (n 4, at least) with strictly controlled temperature were used for a further histopathological assessment. Sham-operated animals (n 4) underwent the same surgical manipulation without occlusion of bilateral common carotid arteries. At 96 h after ischemic insult, animals were anesthetized with pentobarbital and perfused transcardially with saline and then with phosphate-buffered 10% formalin. Brains were removed and processed for paraf®n embedding. Five micrometer coronal sections were cut at the level of the dorsal hippocampus and then stained with hematoxylin and eosin (H&E), followed by TUNEL staining. TUNEL staining was performed to detect the DNA fragmentation of CA1 neurons as described previously [9,10,12,13] with some modi®cation of the method of Gavrieli et al. [7]. The level of nuclear DNA fragmentation and DNA transport in apical dendrites of the CA1 neurons was evaluated using three criteria as described previously [9,10]: (1) nuclear staining; (2) dendritic staining; and (3) dendrite-terminal fragmented DNA pooling. TUNEL staining intensities of (1) nuclear staining and (3) dendrite-terminal fragmented DNA pooling criteria were divided into four grades, negative (2), weakly positive (^), positive (1) and strongly positive (11). The criteria, (2) dendritic staining was graded as follows: grade 0; no staining in the dendrite, grade 1; positive staining within one third of the dendrite, grade 2; positive staining within two thirds of the dendrite, grade 3; positive staining in the whole dendrite. Histological analysis was performed by a blinded observer. The stained sections were examined using a light microscope. Fluorescent dye, DAPI (Boehringer Mannheim GmbH), which intercalates speci®cally into the adenine-thymin base pairs of DNA, was used to identify nuclear DNA and transported DNA in apical dendrites of CA1 neurons [9,10]. The sections were observed using a ¯uorescence microscope.
The level of nuclear DNA fragmentation and DNA transport in apical dendrites of gerbil hippocampal CA1 neurons subjected to 37, 37.5, 38 and 398C during forebrain ischemia has been summarized in Table 1. Strong nuclear TUNEL staining is shown in all CA1 neurons in each temperature (Fig. 1A±D). TUNEL positive cells are limited within CA1 sub®led at 37 and 37.58C. At 388C, scattered TUNEL-positive cells are seen in CA3 and 4 area in addition to CA1 but not in dentate gyrus. Widespread TUNEL-positive cells are recognized in CA3, CA4 sub®leds and dentate gyrus at 398C. Transport of fragmented DNA in whole dendrites of CA1 neurons is apparent at 37 (Fig. 1A), 37.5 (Fig. 1B) and 388C (Fig. 1C), while weak staining is apparent in proximal part of dendrites at 398C (Fig. 1D). Strongly positive staining of dendrite-terminal fragmented DNA pooling is more apparent at 388C (Fig. 1G) than at 378C (Fig. 1E) or at 37.58C (Fig. 1F). Very little staining of fragmented DNA pooling is observable at 398C (Fig. 1H). The localization of fragmented DNA in apical dendrites was con®rmed by DAPI ¯uorescence staining. Since DAPI reactivity was not as strong as that obtained with the TUNEL method, DNA ¯uorescence was not detected in all the apical dendrites in stratum radiatum. However, DNA ¯uorescence was present along the many apical dendrites at the temperatures of 37, 37.5 and 388C (data are not shown). No DAPI positive DNA ¯uorescence was seen along the apical dendrites of hippocampal CA1 regions at 398C (data are not shown). The ¯uorescent intensity of the fragmented DNA pooling in the dendrite-terminal was too weak to be detected by this method, as noted previously [9,10]. Axonal transport delivers organelles, proteins, and other key materials to distant parts of neurons, and requires metabolic energy [6]. Neurons possess a number of unusual Table 1 The level of nuclear DNA fragmentation and DNA transport in apical dendrites of gerbil hippocampal neurons subjected to 37, 37.5, 38 and 398C during forebrain ischemia a Temperature (8C)
Nuclear staining
Dendritic staining
DNA pooling
Sham 37 37.5 38 39
11 11 11 11
0 3 3 3 1
2 1 1 11 ^
a The level of nuclear DNA fragmentation and DNA transport in apical dendrites of the molecular layer of hippocampus was evaluated as three criteria as described previously [9,10]: TUNEL staining intensity of nuclear staining and dendrite-terminal fragmented DNA pooling criteria were divided into four grades, negative (2), weakly positive (^), positive (1) and strongly positive (11). The criteria, dendritic staining was graded as follows: grade 0; no staining in the dendrite, grade 1; positive staining within one third of the dendrite, grade 2; positive staining within two thirds of the dendrite, grade 3; positive staining in the whole dendrite.
A. Hara et al. / Neuroscience Letters 280 (2000) 73±77
75
Fig. 1. Representative microphotographs of apoptotic DNA fragmentation detected by TUNEL staining in hippocampal CA1 sector at 96 h following transient forebrain ischemia in gerbils subjected to 37 (A,E,I), 37.5 (B,F,J), 38 (C,G,K) and 398C (D,H,L) during ischemia. Strong nuclear staining is shown in all neurons of CA1 in each temperature. E, F, G and H show high-power ®eld of rectangles in A, B, C and D, respectively. Transport of fragmented DNA in whole dendrite is recognized in (E,F,G), whereas week staining is observed in proximal part of dendrite in (H). Strongly positive staining of dendrite-terminal fragmented DNA pooling is demonstrated at the temperature of 388C (K), compared with 37 (I) and 37.58C (J). Almost negative staining is observed at the temperature of 398C (L). Scale bars: 425 mm in (A), 50 mm in (E), 33 mm in (I).
properties compared to other cells, due to the fact that dendritic and axonal processes are hundreds or thousands of times longer than the cell body diameter, and that the protein-synthesizing capacity is localized exclusively in the cell body. Consequently, the intracellular transport system plays an essential role in neuronal survival [10]. Recent studies revealed that microtubule-associated motor
molecules such as dynein and kinesin generate the motile force of axonal or dendritic transport with ATP-dependent energy support [14,20]. It is well known that axoplasmic transport is very sensitive to temperature conditions [1,3,16]. The rate of axoplasmic transport increases from 37±428C, and then declines sharply from 42±478C [3]. The temperatures which induce
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A. Hara et al. / Neuroscience Letters 280 (2000) 73±77
most increased velocity of the axoplasmic transport are reported relatively high (40±458C) [1,3,16]. These studies analyzing the dependence of axoplasmic transport on temperature has been performed by using peripheral nerves. The effects of temperature on axoplasmic transport of central nervous system has not been evaluated. Of note, published studies evaluating the effect of hyperthermic temperatures on axoplasmic transport have often utilized temperatures over a very wide, non-physiological range [1,3,16]. For example, Cosens et al. [3] studied the hyperthermic effects at the temperatures of 19, 37, 42 and 478C. In the present study, we histologically demonstrated the temperature-dependent migration of the fragmented DNA from the nuclei into the apical dendrite in the CA1 pyramidal cells following ischemia. Strong nuclear DNA fragmentation was demonstrated in all neurons at the temperatures of 37, 37.5, 38 and 398C. The nuclear DNA fragmentation of CA1 neurons at each temperature was revealed as same level, however DNA transport into apical dendrites and dendrite-terminal fragmented DNA pooling was quite different at the different temperatures. Most pronounced reaction of DNA fragmentation in apical dendrites and fragmented DNA pooling in dendrite-terminal was observed at 388C. In a previous study [11], a 388C temperature during transient forebrain ischemia failed to preserve hippocampal CA1 neurons of gerbil treated with 2-deoxy-D-glucose (2DG; a glucose antimetabolite) although administration of 2DG at 378C or 37.58C effectively prevented the neuronal damage. Apoptotic process is considered to be fairly activated at 388C, compared with 37 or 37.58C. In contrast to the observations at 388C, at 398C there is only weak dendritic staining and almost no staining of fragmented DNA pooling. It is surprising that only a 18C temperature increase makes such a large difference in DNA transport into apical dendrites and dendrite-terminal fragmented DNA pooling. This suggest that there is a temperature threshold of fragmented DNA transport into apical dendrites between 38±398C. Dietrich et al. [4] reported that intraischemic hypothermia at 398C, (1) markedly augmented ischemic brain damage and mortality compared with normothermia, (2) transformed ischemic cell injury into frank infarction, and (3) accelerated the morphological appearance of ischemic brain injury in regions usually demonstrating DND. Minamisawa et al. [17] also showed that a 398C hypothermia signi®cantly enhanced brain damage in several regions, including the hippocampal CA1 region. It is speculated that the 398C temperature is a critical point of hyperthermic damage in ischemic brain tissues. In conclusion, there is a precise temperature dependence for the migration of fragmented DNA from nuclei into apical dendrites of CA1 pyramidal cells during apoptosis following transient forebrain ischemia. The maximal transport of fragmented DNA was observed at 388C. It is hypothesized that 398C temperature is a critical point of
hyperthermic damage in axoplasmic transport although the 388C activates the intracellular metabolism. This work was supported in part by Grant-in-Aid for Encouragement of Young Scientists from the Japanese Ministry of Education, Science, Sports and Culture. The authors wish to thank Ms. Kyoko Takahashi, Chikako Usui and Tomoko Kajita for their technical assistance. [1] Brimijoin, S., Olsen, J. and Rosenson, R., Comparison of the temperature-dependence of rapid axonal transport and microtubules in nerves of the rabbit and bullfrog. J. Physiol., 287 (1979) 303±314. [2] Choi, D.W., Calcium-mediated neurotoxicity: relationship to speci®c channel types and role in ischemic damage. Trends Neurosci., 11 (1988) 465±469. [3] Cosens, B., Thacker, D. and Brimijoin, S., Temperaturedependence of rapid axonal transport in sympathetic nerves of the rabbit. J. Neurobiol., 7 (1976) 339±354. [4] Dietrich, W.D., Busto, R., Valdes, I. and Loor, Y., Effects of normothermic versus mild hyperthermic forebrain ischemia in rats. Stroke, 21 (1990) 1318±1325. [5] Dietrich, W.D., Halley, M., Valdes, I. and Busto, R., Interrelationships between increased vascular permeability and acute neuronal damage following temperature-controlled brain ischemia in rats. Acta Neuropathol. (Berl.), 81 (1991) 615±625. [6] Garcia, A.G., Kirpekar, S.M., Prat, J.C. and Wakade, A.R., Metabolic and ionic requirements for the axoplasmic transport of dopamine-b -hydroxylase. J. Physiol., (1974) 809± 821. [7] Gavrieli, Y., Sherman, Y. and Ben-Sasson, S.A., Identi®cation of programmed cell death in situ via speci®c labeling of nuclear DNA fragmentation. J. Cell Biol., 119 (1992) 493± 501. [8] Globus, M.Y., Busto, R., Lin, B., Schnippering, H. and Ginsberg, M.D., Detection of free radical activity during transient global ischemia and recirculation: effects of intraischemic brain temperature modulation. J. Neurochem., 65 (1995) 1250±1256. [9] Hara, A., Niwa, M., Iwai, T., Nakashima, M., Bunai, Y., Uematsu, T., Yoshimi, N. and Mori, H., Neuronal apoptosis studied by a sequential TUNEL technique: a method for tract-tracing. Brain Res. Prot., 4 (1999) 140±146. [10] Hara, A., Niwa, M., Iwai, T., Nakashima, M., Yano, H., Uematsu, T., Yoshimi, N. and Mori, H., Transport of fragmented DNA in apical dendrites of gerbil CA1 pyramidal neurons following transient forebrain ischemia. Brain Res., 806 (1998) 274±277. [11] Hara, A., Niwa, M., Iwai, T.H., Yano, H., Nakashima, M., Bunai, Y., Uematsu, T., Yoshimi, N. and Mori, H., Failure of preventive effects of 2-deoxy-D-glucose on ischemiainduced gerbil hippocampal neuronal damage by induced hypothermia. Brain Res., 840 (1999) 167±170. [12] Hara, A., Niwa, M., Nakashima, M., Iwai, T., Uematsu, T., Yoshimi, N. and Mori, H., Protective effect of apoptosisinhibitory agent N-tosyl-L-phenylalanyl chloromethyl ketone against ischemia-induced hippocampal neuronal damage. J. Cereb. Blood Flow Metab., 18 (1998) 819±823. [13] Hara, A., Yoshimi, N., Mori, H., Iwai, T., Sakai, N., Yamada, H. and Niwa, M., Hypothermic prevention of nuclear DNA fragmentation in gerbil hippocampus following transient forebrain ischemia. Neurol. Res., 17 (1995) 461±464. [14] Howard, J., Molecular motors: structural adaptations to cellular functions. Nature, 389 (1997) 561±567.
A. Hara et al. / Neuroscience Letters 280 (2000) 73±77 [15] Kil, H.Y., Zhang, J. and Piantadosi, C.A., Brain temperature alters hydroxyl radical production during cerebral ischemia/reperfusion in rats. J. Cereb. Blood Flow Metab., 16 (1996) 100±106. [16] Kim, J.H. and Johnson, J.L., The effects of hypothermia on fast axoplasmic transport in rat sensory ®bers. Brain Res., 237 (1982) 215±221. [17] Minamisawa, H., Smith, M.L. and Siesjo, B.K., The effect of mild hypothermia and hypothermia on brain damage following 5, 10, and 15 min of forebrain ischemia. Ann. Neurol., 28 (1990) 26±33. [18] Nitatori, T., Sato, N., Waguri, S., Karasawa, Y., Araki, H.,
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Shibanai, K., Kominami, E. and Uchiyama, Y., Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis. J. Neurosci., 15 (1995) 1001±1011. [19] Niwa, M., Hara, A., Iwai, T., Nakashima, M., Yano, H., Yoshimi, N., Mori, H. and Uematsu, T., Relationship between magnitude of hypothermia during ischemia and preventive effect against post-ischemic DNA fragmentation in the gerbil hippocampus. Brain Res., 794 (1998) 338±342. [20] Wang, Z., Khan, S. and Sheetz, M.P., Single cytoplasmic dynein molecule movements: Characterization and comparison with kinesin. Biophys. J., 69 (1995) 2011±2023.
Increase of fragmented DNA transport in apical dendrites of gerbil CA1 pyramidal neurons following transient forebrain ischemia by mild hypothermia Akira Hara a,*, Masayuki Niwa b, Tomohiko Iwai c, Hirohito Yano c, Yasuo Bunai d, Toshihiko Uematsu b, Naoki Yoshimi a, Hideki Mori a a Department of Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan Department of Pharmacology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan c Department of Neurosurgery, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan d Department of Legal Medicine, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan b
Received 15 October 1999; received in revised form 2 December 1999; accepted 6 December 1999
Abstract Mild hypothermia (388C) accelerated transport of fragmented DNA in apical dendrites of the gerbil CA1 pyramidal neurons and increased dendrite-terminal fragmented DNA pooling in the apoptotic process following transient forebrain ischemia. The speci®c DNA fragmentation after the ischemic insult in gerbil hippocampus was examined by in situ nickend-labeling method, and ¯uorescence DNA detection technique by DAPI was also performed. There is a precise temperature dependence for the migration of fragmented DNA from nuclei into apical dendrites of CA1 pyramidal cells during apoptosis following transient forebrain ischemia. Increase of fragmented DNA pooling is highly temperature sensitive, occurring at 388C, while at 398C there is a marked decrease in DNA pooling. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Delayed neuronal death; Gerbil; DNA fragmentation; Apical dendrite; Hypothermia
Recently, many studies support the hypothesis that delayed neuronal death (DND) of the CA1 pyramidal neurons after transient forebrain ischemia is not necrotic but apoptotic. Morphological and biochemical data such as DNA ¯uorescence assay, in situ nick-end-labeling method, gel electrophoresis and electron microscope indicate that DND is a form of programmed cell death, or apoptosis. Among them, the most characteristic phenomenon for apoptosis is internucleosomal DNA cleavage into oligonucleosome-length fragments that can be directly visualized by end-labeling with biotinylated dUTP in a reaction that employs terminal deoxynucleotide transferase (TUNEL) [18,19]. In a previous study [10], we histologically demonstrated the migration of the fragmented DNA from the nuclei into the apical dendrite of the CA1 pyramidal neurons of gerbil hippocampus following ischemia, and showed an active transport mechanism of fragmented DNA in the late phase * Corresponding author. Tel.: 181-58-267-2235; fax: 181-58265-9005. E-mail address: [email protected] (A. Hara)
of apoptotic process induced by transient forebrain ischemia. The study focused on not only detection of the nuclear positive reaction of TUNEL, but also on the transportation of fragmented DNA in neuronal ®bers. The phenomenon of the fragmented DNA movement into neuronal ®bers is an evidence of intracellular metabolic mechanisms that occur during the apoptotic process. Hypothermia during transient forebrain ischemia exacerbates ischemia-induced neuronal death in hippocampal CA1 neurons [4,11,15]. Production of reactive oxygen species [15], hydroxyl free radicals [8], calcium in¯ux into neurons [2] and vascular permeability [5] have been reported to be enhanced by hypothermia in neurons of hippocampal CA1 neurons. It is also well known that axoplasmic transportation is very sensitive to temperature conditions [16]. Average transport velocity is exponentially related to temperature over a wide range, although most studies of axoplasmic transport have been performed by using peripheral nerves. There are no reports evaluating the effects of hypothermia on the axoplasmic transportation within the central nervous system.
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 97 9- 9
74
A. Hara et al. / Neuroscience Letters 280 (2000) 73±77
In the present study, using the transient forebrain ischemia model for apoptosis, we evaluated in gerbil CA1 pyramidal neurons the effects of temperatures (37±398C) on fragmented DNA movement (in neuronal ®bers) and pooling (in dendrite terminals). Male Mongolian gerbils, weighing 65±75 g, were subjected to severe forebrain ischemia as described previously [11±13]. Anesthesia was induced with 4% halothane and maintained with 2% halothane during subsequent surgery. Carotid arteries were isolated through an anterior midcervical incision, and animals were subjected to 5 min forebrain ischemia with microclips applied to the carotid arteries. Rectal temperatures of 37, 37.5, 38 or 398C, maintained using a heating pad with a circulating system, were adjusted just before, during and just after ischemic insult. After reperfusion, the temperature was maintained at 378C for 3 h. The rectal temperature was monitored and recorded continuously. If the recording showed that body temperature was not maintained at the pre-de®ned temperature, the animal was excluded from the study. Accordingly, groups of animals (n 4, at least) with strictly controlled temperature were used for a further histopathological assessment. Sham-operated animals (n 4) underwent the same surgical manipulation without occlusion of bilateral common carotid arteries. At 96 h after ischemic insult, animals were anesthetized with pentobarbital and perfused transcardially with saline and then with phosphate-buffered 10% formalin. Brains were removed and processed for paraf®n embedding. Five micrometer coronal sections were cut at the level of the dorsal hippocampus and then stained with hematoxylin and eosin (H&E), followed by TUNEL staining. TUNEL staining was performed to detect the DNA fragmentation of CA1 neurons as described previously [9,10,12,13] with some modi®cation of the method of Gavrieli et al. [7]. The level of nuclear DNA fragmentation and DNA transport in apical dendrites of the CA1 neurons was evaluated using three criteria as described previously [9,10]: (1) nuclear staining; (2) dendritic staining; and (3) dendrite-terminal fragmented DNA pooling. TUNEL staining intensities of (1) nuclear staining and (3) dendrite-terminal fragmented DNA pooling criteria were divided into four grades, negative (2), weakly positive (^), positive (1) and strongly positive (11). The criteria, (2) dendritic staining was graded as follows: grade 0; no staining in the dendrite, grade 1; positive staining within one third of the dendrite, grade 2; positive staining within two thirds of the dendrite, grade 3; positive staining in the whole dendrite. Histological analysis was performed by a blinded observer. The stained sections were examined using a light microscope. Fluorescent dye, DAPI (Boehringer Mannheim GmbH), which intercalates speci®cally into the adenine-thymin base pairs of DNA, was used to identify nuclear DNA and transported DNA in apical dendrites of CA1 neurons [9,10]. The sections were observed using a ¯uorescence microscope.
The level of nuclear DNA fragmentation and DNA transport in apical dendrites of gerbil hippocampal CA1 neurons subjected to 37, 37.5, 38 and 398C during forebrain ischemia has been summarized in Table 1. Strong nuclear TUNEL staining is shown in all CA1 neurons in each temperature (Fig. 1A±D). TUNEL positive cells are limited within CA1 sub®led at 37 and 37.58C. At 388C, scattered TUNEL-positive cells are seen in CA3 and 4 area in addition to CA1 but not in dentate gyrus. Widespread TUNEL-positive cells are recognized in CA3, CA4 sub®leds and dentate gyrus at 398C. Transport of fragmented DNA in whole dendrites of CA1 neurons is apparent at 37 (Fig. 1A), 37.5 (Fig. 1B) and 388C (Fig. 1C), while weak staining is apparent in proximal part of dendrites at 398C (Fig. 1D). Strongly positive staining of dendrite-terminal fragmented DNA pooling is more apparent at 388C (Fig. 1G) than at 378C (Fig. 1E) or at 37.58C (Fig. 1F). Very little staining of fragmented DNA pooling is observable at 398C (Fig. 1H). The localization of fragmented DNA in apical dendrites was con®rmed by DAPI ¯uorescence staining. Since DAPI reactivity was not as strong as that obtained with the TUNEL method, DNA ¯uorescence was not detected in all the apical dendrites in stratum radiatum. However, DNA ¯uorescence was present along the many apical dendrites at the temperatures of 37, 37.5 and 388C (data are not shown). No DAPI positive DNA ¯uorescence was seen along the apical dendrites of hippocampal CA1 regions at 398C (data are not shown). The ¯uorescent intensity of the fragmented DNA pooling in the dendrite-terminal was too weak to be detected by this method, as noted previously [9,10]. Axonal transport delivers organelles, proteins, and other key materials to distant parts of neurons, and requires metabolic energy [6]. Neurons possess a number of unusual Table 1 The level of nuclear DNA fragmentation and DNA transport in apical dendrites of gerbil hippocampal neurons subjected to 37, 37.5, 38 and 398C during forebrain ischemia a Temperature (8C)
Nuclear staining
Dendritic staining
DNA pooling
Sham 37 37.5 38 39
11 11 11 11
0 3 3 3 1
2 1 1 11 ^
a The level of nuclear DNA fragmentation and DNA transport in apical dendrites of the molecular layer of hippocampus was evaluated as three criteria as described previously [9,10]: TUNEL staining intensity of nuclear staining and dendrite-terminal fragmented DNA pooling criteria were divided into four grades, negative (2), weakly positive (^), positive (1) and strongly positive (11). The criteria, dendritic staining was graded as follows: grade 0; no staining in the dendrite, grade 1; positive staining within one third of the dendrite, grade 2; positive staining within two thirds of the dendrite, grade 3; positive staining in the whole dendrite.
A. Hara et al. / Neuroscience Letters 280 (2000) 73±77
75
Fig. 1. Representative microphotographs of apoptotic DNA fragmentation detected by TUNEL staining in hippocampal CA1 sector at 96 h following transient forebrain ischemia in gerbils subjected to 37 (A,E,I), 37.5 (B,F,J), 38 (C,G,K) and 398C (D,H,L) during ischemia. Strong nuclear staining is shown in all neurons of CA1 in each temperature. E, F, G and H show high-power ®eld of rectangles in A, B, C and D, respectively. Transport of fragmented DNA in whole dendrite is recognized in (E,F,G), whereas week staining is observed in proximal part of dendrite in (H). Strongly positive staining of dendrite-terminal fragmented DNA pooling is demonstrated at the temperature of 388C (K), compared with 37 (I) and 37.58C (J). Almost negative staining is observed at the temperature of 398C (L). Scale bars: 425 mm in (A), 50 mm in (E), 33 mm in (I).
properties compared to other cells, due to the fact that dendritic and axonal processes are hundreds or thousands of times longer than the cell body diameter, and that the protein-synthesizing capacity is localized exclusively in the cell body. Consequently, the intracellular transport system plays an essential role in neuronal survival [10]. Recent studies revealed that microtubule-associated motor
molecules such as dynein and kinesin generate the motile force of axonal or dendritic transport with ATP-dependent energy support [14,20]. It is well known that axoplasmic transport is very sensitive to temperature conditions [1,3,16]. The rate of axoplasmic transport increases from 37±428C, and then declines sharply from 42±478C [3]. The temperatures which induce
76
A. Hara et al. / Neuroscience Letters 280 (2000) 73±77
most increased velocity of the axoplasmic transport are reported relatively high (40±458C) [1,3,16]. These studies analyzing the dependence of axoplasmic transport on temperature has been performed by using peripheral nerves. The effects of temperature on axoplasmic transport of central nervous system has not been evaluated. Of note, published studies evaluating the effect of hyperthermic temperatures on axoplasmic transport have often utilized temperatures over a very wide, non-physiological range [1,3,16]. For example, Cosens et al. [3] studied the hyperthermic effects at the temperatures of 19, 37, 42 and 478C. In the present study, we histologically demonstrated the temperature-dependent migration of the fragmented DNA from the nuclei into the apical dendrite in the CA1 pyramidal cells following ischemia. Strong nuclear DNA fragmentation was demonstrated in all neurons at the temperatures of 37, 37.5, 38 and 398C. The nuclear DNA fragmentation of CA1 neurons at each temperature was revealed as same level, however DNA transport into apical dendrites and dendrite-terminal fragmented DNA pooling was quite different at the different temperatures. Most pronounced reaction of DNA fragmentation in apical dendrites and fragmented DNA pooling in dendrite-terminal was observed at 388C. In a previous study [11], a 388C temperature during transient forebrain ischemia failed to preserve hippocampal CA1 neurons of gerbil treated with 2-deoxy-D-glucose (2DG; a glucose antimetabolite) although administration of 2DG at 378C or 37.58C effectively prevented the neuronal damage. Apoptotic process is considered to be fairly activated at 388C, compared with 37 or 37.58C. In contrast to the observations at 388C, at 398C there is only weak dendritic staining and almost no staining of fragmented DNA pooling. It is surprising that only a 18C temperature increase makes such a large difference in DNA transport into apical dendrites and dendrite-terminal fragmented DNA pooling. This suggest that there is a temperature threshold of fragmented DNA transport into apical dendrites between 38±398C. Dietrich et al. [4] reported that intraischemic hypothermia at 398C, (1) markedly augmented ischemic brain damage and mortality compared with normothermia, (2) transformed ischemic cell injury into frank infarction, and (3) accelerated the morphological appearance of ischemic brain injury in regions usually demonstrating DND. Minamisawa et al. [17] also showed that a 398C hypothermia signi®cantly enhanced brain damage in several regions, including the hippocampal CA1 region. It is speculated that the 398C temperature is a critical point of hyperthermic damage in ischemic brain tissues. In conclusion, there is a precise temperature dependence for the migration of fragmented DNA from nuclei into apical dendrites of CA1 pyramidal cells during apoptosis following transient forebrain ischemia. The maximal transport of fragmented DNA was observed at 388C. It is hypothesized that 398C temperature is a critical point of
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