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Selective vulnerability to radiation in the hippocampal dentate granule cells
Selective Vulnerability to Radiation in the Hippocampal Dentate Granule Cells Rie Nagai, M.Sc., Shigeru Tsunoda, M.D., Ph.D., Yasuharu Hori, Ph.D., and Hiroshi Asada, Ph.D. Laboratory of Neuropathology, Graduate School of Science and Laboratory of Radiation Biology, Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, and Department of Neurosurgery, Nara Medical University, Kashihara, Japan
Nagai R, Tsunoda S, Hori Y, Asada H. Selective vulnerability to radiation in the hippocampal dentate granule cells. Surg Neurol 2000;53:503– 07.
KEY WORDS
Radiation, apoptosis, cerebrum, hippocampus, dentate granule cells.
BACKGROUND
Radiation therapy is an effective approach in the treatment of highly radiosensitive brain tumors such as germinomas. However, recent studies have reported intellectual disturbances in patients who underwent wholebrain irradiation as children. We detected apoptosis in the infantile murine cerebrum after systemic X-ray irradiation. METHODS
Subjects were 100 ICR mice 4 weeks old, of which 90 were systemically exposed to 18 Gy X-rays (0.45 Gy/min); 10 each were decapitated and the cerebrums were removed 1, 3, 6, 9, 12, 18, 24, 48, and 72 hours after irradiation. Controls were 10 unirradiated mice. DNA fragmentation analysis was carried out by agarose gel electrophoresis, and morphological analysis was by the TUNEL method. RESULTS
According to agarose gel electrophoresis, the cerebral DNA ladders were detected only over 6 to 24 hr, peaking in 9 hr. Even at the peak, band intensity was nearly double that of the unirradiated normal thymus. According to the TUNEL analysis, radiation-induced apoptosis increased, with a peak at 9 hours, but decreased 24 hours after irradiation. Apoptotic cells were always localized exclusively in the hippocampal dentate granule cells. CONCLUSIONS
We found that vulnerability to radiation existed in the hippocampal dentate granule cells. Intellectual disturbances in patients who have undergone whole-brain irradiation may be caused by injury to the hippocampus. © 2000 by Elsevier Science Inc.
Address reprint requests to: Dr Shigeru Tsunoda, Laboratory of Neuropathology, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai 599-8531, Japan Received November 24, 1999; accepted February 18, 2000. © 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
adiation therapy is an effective approach in the treatment of highly radiosensitive brain tumors such as germinomas. However, recent studies have reported intellectual disturbances in patients who underwent whole-brain irradiation as children [1,5,6]. And many studies of prenatally exposed survivors of the atomic bombings of Hiroshima and Nagasaki have shown that exposure to ionizing radiation during gestation has harmful effects on the developing human brain [19]. Radiation-induced cell death was considered to be necrosis, but recent studies show involvement of apoptosis [16,17,21]. However, we found few reports on radiation-induced apoptosis in the cerebrum [13], and no reports on selective vulnerability to radiation. We detected apoptosis in the infantile murine cerebrum after systemic X-ray irradiation.
R
Materials and Methods Subjects were 100 ICR mice 4 weeks old, of which 90 were systemically exposed to 18 Gy X-rays (0.45 Gy/ min); 10 each were decapitated and the cerebrums were removed 1, 3, 6, 9, 12, 18, 24, 48, and 72 hr after irradiation. This dosage of irradiation was chosen according to the data from our preliminary experiments. Less than 10 Gy causes no abnormal apoptosis in the cerebrum, but more than 20 Gy provokes radiationinduced apoptosis not only in the hippocampus but also in the cerebral cortex. On the basis of these data, we set the dosage at 18 Gy. Controls were 10 unirra0090-3019/00/$–see front matter PII S0090-3019(00)00214-7
504 Surg Neurol 2000;53:503–07
Nagai et al
diated mice. Fifty cerebrums from each group were frozen and stored at ⫺80°C for DNA fragmentation analysis. Others were fixed in 10% neutral buffered formalin, embedded in paraffin, and then cut into 3 m sections for morphological study. DNA FRAGMENTATION ANALYSIS Cells were lysed in lysis buffer (0.1% sodium dodecyl sulfate, 10 mM Tris, 10 mM EDTA; pH 8.0) containing 10 g/ml of proteinase K. The mixture was incubated at 55°C for 120 min. Cellular DNA was extracted with phenol and precipitated with ethanol. Precipitated DNA was resuspended in TE buffer (10 mM Tris, 10 mM EDTA: pH 8.0). The DNA samples were subjected to electrophoresis in 2% agarose gel and stained with ethidium bromide to detect fluorescence by an ultraviolet (UV) transilluminator. To compare differences in DNA fragmentation, fluorescent DNA bands were scanned as digital images by a computer using imaging software developed by NIH. MORPHOLOGICAL ANALYSIS For the detection of DNA fragmentation in situ using the TUNEL method [8], cells were processed according to the instructions in the ApopTag kit (Oncor, Gaithersburg, MD, USA). In brief, digoxigenin-deoxyuridine 5-phosphate was used to label the 3⬘-OH ends by terminal deoxynucleotidyltransferase, and digoxigenin was detected by antidigoxigenin-peroxidase labeling and then by 3, 3⬘-diaminobenzidine HCl reaction. As a positive control, we pretreated slides with DNase I (Stratagene, La Jolla, CA, USA) to produce TUNELpositive staining of all nuclei, and the negative controls were incubated without TdT enzyme.
Agarose gel electrophoresis of cerebral DNA. The cerebral DNA ladders using 10 g of DNA were detected only over 6 to 24 hr, peaking in 9 hr. (T: unirradiated thymus using 10 g of DNA, C: unirradiated cerebrum).
1
Discussion The hippocampus is known to have selective vulnerabilities to various harmful stimuli. In particular, the hippocampal CA1 pyramidal neurons are easily damaged by global cerebral ischemia [2,15] and are known to show evidence of apoptotic cell death [4,11,14]. On the other hand, long-term adrenalectomy (ADX) is reported to result in apoptosis within the dentate gyrus of the mature rat hippocampus [20,22,23]. The mechanisms underlying this cell
Results Agarose gel electrophoresis was carried out for DNA fragmentation analysis. The cerebral DNA ladders detected only over 6 to 24 hr, peaking in 9 hr (Figure 1). Even at the peak, however, band intensity was nearly double that of the unirradiated normal thymus (Figure 2). TUNEL method was carried out for morphological analysis. In the cerebrum, we could detect few apoptotic cells in the hippocampal dentate granule cells of unirradiated controls (Figure 3-A). The apoptotic cells increased in number, with a peak at 9 hr, but decreased in number 24 hr after irradiation. Apoptotic cells were always localized exclusively in the hippocampal dentate granule cells (Figure 3-C). No apoptotic cells were detected in any other regions of the cerebrum before or after irradiation (Figure 3-B, D).
Comparison of band intensity in cerebral DNA ladders. Band intensities were calculated in all groups in comparison to controls (unirradiated thymuses) evaluated as 1. The DNA ladders could be seen only over 6 to 24 hr. ✻p ⬍ 0.01, ✻✻p ⬍ 0.05, compared with controls (Mann–Whitney’s test).
2
Radiation-Induced Hippocampal Apoptosis
Surg Neurol 505 2000;53:503–07
Photograms of the cerebrum. A. The unirradiated hippocampus. There are hardly any apoptotic cells among the hippocampal dentate granule cells (TUNEL method, ⫻50). B. The unirradiated cerebral cortex. No apoptotic cells are detected in the cerebral cortex (TUNEL method, ⫻50). C. The hippocampus 9 hr after irradiation. Apoptotic cells are detected exclusively among the hippocampal dentate granule cells after irradiation (TUNEL method, ⫻50). D. The cerebral cortex 9 hr after irradiation. No apoptotic cells are detected in the cerebral cortex even after irradiation (TUNEL method, ⫻50).
3
death have not been fully clarified. It is now considered that the granule cells require adrenal steroids for their survival, since corticosterone replacement prevents their death [3,9,12]. The cerebrum has lower radiosensitivity than any other organ [24,25]. We detected apoptosis exclusively in the dentate gyrus of the hippocampus after systemic 18 Gy irradiation. We found that vulnerability to radiation existed in the hippocampal dentate granule cells similar to ADX-induced apoptosis. We could not detect any apoptosis in the hippocampal CA1 pyramidal neurons in this experiment, though the neurons of CA1 usually react to cerebral ischemia by expressing certain early genes [7,10,18]. The granule cells of the rat dentate gyrus continue to form in adulthood as well as during early development. The proliferation potential of the hippocampal dentate granule cell is known to be higher than any other part of the cerebrum [3].
The selective vulnerability to radiation is thought to relate to the proliferation potential of the hippocampal dentate granule cells. We are now investigating the etiology of this selective vulnerability from the viewpoint of molecular biology. Recent physiological studies on memory have shown that the hippocampus plays a significant role in short-term memory. Intellectual disturbances in patients who have undergone wholebrain irradiation may be caused by injury to the hippocampus. Experiments in space flight are becoming increasingly common. The effects of cosmic rays on astronauts should be studied because of the possibility of long-term exposure in space causing dementia as a result of hippocampal damage. This work is supported in part by a Grant-in-Aid for Scientific Research (C: 09671441) for the Ministry of Education, Science and Culture, Japan.
506 Surg Neurol 2000;53:503–07
Nagai et al
REFERENCES 1. Al–Mefty O, Kersh JE, Routh A, Smith RR. The longterm side effects of radiation therapy for benign brain tumors in adults. J Neurosurg 1990;73:502–12. 2. Abe K, Aoki M, Kawagoe J, et al. Ischemic delayed neuronal death. A mitochondrial hypothesis. Stroke 1995;26:1478 – 89. 3. Cameron HA, Gould E. Distinct populations of cells in the adult dentate gyrus undergo mitosis or apoptosis in response to adrenalectomy. J Comp Neurol 1996; 369:56 – 63. 4. Chen J, Zhu RL, Nakayama M, et al. Expression of the apoptosis-effector gene, Bax, is up-regulated in vulnerable hippocampal CA1 neurons after global ischemia. J Neurochem 1996;67:64 –71. 5. Danoff BF, Cowchock FS, Marquette C, Mulgrew L, Kramer S. Assessment of the long-term effects of primary radiation therapy for brain tumors in children. Cancer 1982;49:1580 – 86. 6. Dennis M, Spiegler BJ, Hetherington CR, Greenberg ML. Neuropsychological sequelae of the treatment of children with medulloblastoma. J Neurooncol 1996; 29:91–101. 7. Dragunow M, Faull RLM, Jansen KLR. MK-801, an antagonist of NMDA receptors, inhibits injury-induced c-fos protein accumulation in rat brain. Neurosci Lett 1990;109:128 –33. 8. Gavrieli Y, Sherman Y, Ben–Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119: 493–501. 9. Gerth A, Hatalski CG, Avishai–Eliner S, Baram TZ. Corticotropin releasing hormone antagonist does not prevent adrenalectomy-induced apoptosis in the dentate gyrus of the rat hippocampus. Stress 1998;2:159 – 69. 10. Ham J, Babij C, Whitfield J, et al. A c-jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron 1995;14:927– 39. 11. Honkaniemi J, Massa SM, Breckinridge M, Sharp FR. Global ischemia induces apoptosis-associated genes in hippocampus. Brain Res Mol Brain Res 1996;42:79 – 88. 12. Hornsby CD, Grootendorst J, de Kloet ER. Dexamethasone does not prevent 7-day ADX-induced apoptosis in the dentate gyrus of the rat hippocampus. Stress 1996;1:51– 64. 13. Johnson MD, Xiang H, London S, et al. Evidence for involvement of Bax and p53, but not caspases, in radiation-induced cell death of cultured postnatal hippocampal neurons. J Neurosci Res 1998;54:721– 33. 14. Kihara S, Shiraishi T, Nakagawa S, Toda K, Tabuchi K. Visualization of DNA double strand breaks in the gerbil hippocampal CA1 after transient ischemia. Neurosci Lett 1994;175:133– 6. 15. Kirino T. Delayed neuronal death in the gerbil hippocampus after ischemia. Brain Res 1982;239:57– 69. 16. Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 1993;362:847–9. 17. Maki CG, Howley PM. Ubiquitination of p53 and p21 is
18. 19.
20. 21. 22.
23.
24. 25.
differentially affected by ionizing and UV radiation. Mol Cell Biol 1997;17:355– 63. Nagata S. Apoptosis by death factor. Cell 1997;88:355– 65. Otake M, Schull WJ. Radiation-related brain damage and growth retardation among the prenatally exposed atomic bomb survivors. Int J Radiat Biol 1998; 74:159 –71. Sakhi S, Gilmore W, Tran ND, Schreiber SS. p53deficient mice are protected against adrenalectomyinduced apoptosis. Neuroreport 1996;8:233–5. Savitz SI, Rosenbaum DM. Apoptosis in neurological disease. Neurosurgery 1998;42:555–74. Sloviter RS, Sollas AL, Dean E, Neubort S. Adrenalectomy-induced granule cell degeneration in the rat hippocampal dentate gyrus: characterization of an in vivo model of controlled neuronal death. J Comp Neurol 1993;330:324 –36. Sloviter RS, Sollas AL, Neubort S. Hippocampal dentate granule cell degeneration after adrenalectomy in the rat is not reversed by dexamethasone. Brain Res 1995;682:227–30. Suciu D. Morphometric study of the interphase nucleus in some radiosensitive and radioresistant mammalian cells. J Theor Biol 1985;113:599 – 609. Zhang H, Wheeler KT. Radiation-induced DNA damage in tumors and normal tissues. Radiat Res 1994; 140:321– 6.
COMMENTARY
Because radiation treatment has become a standard procedure in the management of brain tumors, the issue addressed in this paper is of high clinical relevance. For the first time, the authors have shown significant vulnerability of dentate granule cells in the infant mouse after exposure to a single dose of 18 Gy. A few hours after irradiation, apoptosis was found in the dentate granule cells, but not in other areas of the brain. This is an important contribution to our understanding of the well-known neuropsychological deficits caused by fractionated whole-brain radiation therapy, although single-dose irradiation might have different molecular biological effects and the conditions in humans may be different from those in rodents. Despite these restrictions, the reported results could have considerable impact on the therapeutic strategies in neuro-oncology, stressing the importance of focal irradiation (conformal stereotactic RT, brachytherapy, radiosurgery), which has a much higher potential for sparing the sensitive hippocampal structures than conventional radiation methods. Prof. Dr. med. Volker Sturm Department of Stereotactic and Functional Neurosurgery University of Cologne Cologne, Germany
Radiation-Induced Hippocampal Apoptosis
Surg Neurol 507 2000;53:503–07
This study assesses a possible mechanism of mental retardation: whole-brain radiation for brain tumors in infants and children. The dose given of 18 Gy at one shot to infantile murine whole cerebral seems to be very high compared to most clinical cases. It has already been noted that high-dose radiation induces apoptosis of cells, but vulnerability of neuronal cells to radiation in different areas of the brain has not been
clarified. Therefore, this work, which demonstrates the difference, is a valuable resource and may help prevent intellectual disturbances in children who receive radiotherapy. Kintomo Takakura, M.D., Ph.D. Department of Neurosurgery Tokyo Women’s Medical University Tokyo, Japan
orrie was able to joke about his body now. The closer he got to the end, the more he saw it as a mere shell, a container of the soul. It was withering to useless skin and bones anyhow, which made it easier to let go.
M
—Mitch Albom, “Tuesdays with Morrie”
Nagai R, Tsunoda S, Hori Y, Asada H. Selective vulnerability to radiation in the hippocampal dentate granule cells. Surg Neurol 2000;53:503– 07.
KEY WORDS
Radiation, apoptosis, cerebrum, hippocampus, dentate granule cells.
BACKGROUND
Radiation therapy is an effective approach in the treatment of highly radiosensitive brain tumors such as germinomas. However, recent studies have reported intellectual disturbances in patients who underwent wholebrain irradiation as children. We detected apoptosis in the infantile murine cerebrum after systemic X-ray irradiation. METHODS
Subjects were 100 ICR mice 4 weeks old, of which 90 were systemically exposed to 18 Gy X-rays (0.45 Gy/min); 10 each were decapitated and the cerebrums were removed 1, 3, 6, 9, 12, 18, 24, 48, and 72 hours after irradiation. Controls were 10 unirradiated mice. DNA fragmentation analysis was carried out by agarose gel electrophoresis, and morphological analysis was by the TUNEL method. RESULTS
According to agarose gel electrophoresis, the cerebral DNA ladders were detected only over 6 to 24 hr, peaking in 9 hr. Even at the peak, band intensity was nearly double that of the unirradiated normal thymus. According to the TUNEL analysis, radiation-induced apoptosis increased, with a peak at 9 hours, but decreased 24 hours after irradiation. Apoptotic cells were always localized exclusively in the hippocampal dentate granule cells. CONCLUSIONS
We found that vulnerability to radiation existed in the hippocampal dentate granule cells. Intellectual disturbances in patients who have undergone whole-brain irradiation may be caused by injury to the hippocampus. © 2000 by Elsevier Science Inc.
Address reprint requests to: Dr Shigeru Tsunoda, Laboratory of Neuropathology, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai 599-8531, Japan Received November 24, 1999; accepted February 18, 2000. © 2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
adiation therapy is an effective approach in the treatment of highly radiosensitive brain tumors such as germinomas. However, recent studies have reported intellectual disturbances in patients who underwent whole-brain irradiation as children [1,5,6]. And many studies of prenatally exposed survivors of the atomic bombings of Hiroshima and Nagasaki have shown that exposure to ionizing radiation during gestation has harmful effects on the developing human brain [19]. Radiation-induced cell death was considered to be necrosis, but recent studies show involvement of apoptosis [16,17,21]. However, we found few reports on radiation-induced apoptosis in the cerebrum [13], and no reports on selective vulnerability to radiation. We detected apoptosis in the infantile murine cerebrum after systemic X-ray irradiation.
R
Materials and Methods Subjects were 100 ICR mice 4 weeks old, of which 90 were systemically exposed to 18 Gy X-rays (0.45 Gy/ min); 10 each were decapitated and the cerebrums were removed 1, 3, 6, 9, 12, 18, 24, 48, and 72 hr after irradiation. This dosage of irradiation was chosen according to the data from our preliminary experiments. Less than 10 Gy causes no abnormal apoptosis in the cerebrum, but more than 20 Gy provokes radiationinduced apoptosis not only in the hippocampus but also in the cerebral cortex. On the basis of these data, we set the dosage at 18 Gy. Controls were 10 unirra0090-3019/00/$–see front matter PII S0090-3019(00)00214-7
504 Surg Neurol 2000;53:503–07
Nagai et al
diated mice. Fifty cerebrums from each group were frozen and stored at ⫺80°C for DNA fragmentation analysis. Others were fixed in 10% neutral buffered formalin, embedded in paraffin, and then cut into 3 m sections for morphological study. DNA FRAGMENTATION ANALYSIS Cells were lysed in lysis buffer (0.1% sodium dodecyl sulfate, 10 mM Tris, 10 mM EDTA; pH 8.0) containing 10 g/ml of proteinase K. The mixture was incubated at 55°C for 120 min. Cellular DNA was extracted with phenol and precipitated with ethanol. Precipitated DNA was resuspended in TE buffer (10 mM Tris, 10 mM EDTA: pH 8.0). The DNA samples were subjected to electrophoresis in 2% agarose gel and stained with ethidium bromide to detect fluorescence by an ultraviolet (UV) transilluminator. To compare differences in DNA fragmentation, fluorescent DNA bands were scanned as digital images by a computer using imaging software developed by NIH. MORPHOLOGICAL ANALYSIS For the detection of DNA fragmentation in situ using the TUNEL method [8], cells were processed according to the instructions in the ApopTag kit (Oncor, Gaithersburg, MD, USA). In brief, digoxigenin-deoxyuridine 5-phosphate was used to label the 3⬘-OH ends by terminal deoxynucleotidyltransferase, and digoxigenin was detected by antidigoxigenin-peroxidase labeling and then by 3, 3⬘-diaminobenzidine HCl reaction. As a positive control, we pretreated slides with DNase I (Stratagene, La Jolla, CA, USA) to produce TUNELpositive staining of all nuclei, and the negative controls were incubated without TdT enzyme.
Agarose gel electrophoresis of cerebral DNA. The cerebral DNA ladders using 10 g of DNA were detected only over 6 to 24 hr, peaking in 9 hr. (T: unirradiated thymus using 10 g of DNA, C: unirradiated cerebrum).
1
Discussion The hippocampus is known to have selective vulnerabilities to various harmful stimuli. In particular, the hippocampal CA1 pyramidal neurons are easily damaged by global cerebral ischemia [2,15] and are known to show evidence of apoptotic cell death [4,11,14]. On the other hand, long-term adrenalectomy (ADX) is reported to result in apoptosis within the dentate gyrus of the mature rat hippocampus [20,22,23]. The mechanisms underlying this cell
Results Agarose gel electrophoresis was carried out for DNA fragmentation analysis. The cerebral DNA ladders detected only over 6 to 24 hr, peaking in 9 hr (Figure 1). Even at the peak, however, band intensity was nearly double that of the unirradiated normal thymus (Figure 2). TUNEL method was carried out for morphological analysis. In the cerebrum, we could detect few apoptotic cells in the hippocampal dentate granule cells of unirradiated controls (Figure 3-A). The apoptotic cells increased in number, with a peak at 9 hr, but decreased in number 24 hr after irradiation. Apoptotic cells were always localized exclusively in the hippocampal dentate granule cells (Figure 3-C). No apoptotic cells were detected in any other regions of the cerebrum before or after irradiation (Figure 3-B, D).
Comparison of band intensity in cerebral DNA ladders. Band intensities were calculated in all groups in comparison to controls (unirradiated thymuses) evaluated as 1. The DNA ladders could be seen only over 6 to 24 hr. ✻p ⬍ 0.01, ✻✻p ⬍ 0.05, compared with controls (Mann–Whitney’s test).
2
Radiation-Induced Hippocampal Apoptosis
Surg Neurol 505 2000;53:503–07
Photograms of the cerebrum. A. The unirradiated hippocampus. There are hardly any apoptotic cells among the hippocampal dentate granule cells (TUNEL method, ⫻50). B. The unirradiated cerebral cortex. No apoptotic cells are detected in the cerebral cortex (TUNEL method, ⫻50). C. The hippocampus 9 hr after irradiation. Apoptotic cells are detected exclusively among the hippocampal dentate granule cells after irradiation (TUNEL method, ⫻50). D. The cerebral cortex 9 hr after irradiation. No apoptotic cells are detected in the cerebral cortex even after irradiation (TUNEL method, ⫻50).
3
death have not been fully clarified. It is now considered that the granule cells require adrenal steroids for their survival, since corticosterone replacement prevents their death [3,9,12]. The cerebrum has lower radiosensitivity than any other organ [24,25]. We detected apoptosis exclusively in the dentate gyrus of the hippocampus after systemic 18 Gy irradiation. We found that vulnerability to radiation existed in the hippocampal dentate granule cells similar to ADX-induced apoptosis. We could not detect any apoptosis in the hippocampal CA1 pyramidal neurons in this experiment, though the neurons of CA1 usually react to cerebral ischemia by expressing certain early genes [7,10,18]. The granule cells of the rat dentate gyrus continue to form in adulthood as well as during early development. The proliferation potential of the hippocampal dentate granule cell is known to be higher than any other part of the cerebrum [3].
The selective vulnerability to radiation is thought to relate to the proliferation potential of the hippocampal dentate granule cells. We are now investigating the etiology of this selective vulnerability from the viewpoint of molecular biology. Recent physiological studies on memory have shown that the hippocampus plays a significant role in short-term memory. Intellectual disturbances in patients who have undergone wholebrain irradiation may be caused by injury to the hippocampus. Experiments in space flight are becoming increasingly common. The effects of cosmic rays on astronauts should be studied because of the possibility of long-term exposure in space causing dementia as a result of hippocampal damage. This work is supported in part by a Grant-in-Aid for Scientific Research (C: 09671441) for the Ministry of Education, Science and Culture, Japan.
506 Surg Neurol 2000;53:503–07
Nagai et al
REFERENCES 1. Al–Mefty O, Kersh JE, Routh A, Smith RR. The longterm side effects of radiation therapy for benign brain tumors in adults. J Neurosurg 1990;73:502–12. 2. Abe K, Aoki M, Kawagoe J, et al. Ischemic delayed neuronal death. A mitochondrial hypothesis. Stroke 1995;26:1478 – 89. 3. Cameron HA, Gould E. Distinct populations of cells in the adult dentate gyrus undergo mitosis or apoptosis in response to adrenalectomy. J Comp Neurol 1996; 369:56 – 63. 4. Chen J, Zhu RL, Nakayama M, et al. Expression of the apoptosis-effector gene, Bax, is up-regulated in vulnerable hippocampal CA1 neurons after global ischemia. J Neurochem 1996;67:64 –71. 5. Danoff BF, Cowchock FS, Marquette C, Mulgrew L, Kramer S. Assessment of the long-term effects of primary radiation therapy for brain tumors in children. Cancer 1982;49:1580 – 86. 6. Dennis M, Spiegler BJ, Hetherington CR, Greenberg ML. Neuropsychological sequelae of the treatment of children with medulloblastoma. J Neurooncol 1996; 29:91–101. 7. Dragunow M, Faull RLM, Jansen KLR. MK-801, an antagonist of NMDA receptors, inhibits injury-induced c-fos protein accumulation in rat brain. Neurosci Lett 1990;109:128 –33. 8. Gavrieli Y, Sherman Y, Ben–Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119: 493–501. 9. Gerth A, Hatalski CG, Avishai–Eliner S, Baram TZ. Corticotropin releasing hormone antagonist does not prevent adrenalectomy-induced apoptosis in the dentate gyrus of the rat hippocampus. Stress 1998;2:159 – 69. 10. Ham J, Babij C, Whitfield J, et al. A c-jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron 1995;14:927– 39. 11. Honkaniemi J, Massa SM, Breckinridge M, Sharp FR. Global ischemia induces apoptosis-associated genes in hippocampus. Brain Res Mol Brain Res 1996;42:79 – 88. 12. Hornsby CD, Grootendorst J, de Kloet ER. Dexamethasone does not prevent 7-day ADX-induced apoptosis in the dentate gyrus of the rat hippocampus. Stress 1996;1:51– 64. 13. Johnson MD, Xiang H, London S, et al. Evidence for involvement of Bax and p53, but not caspases, in radiation-induced cell death of cultured postnatal hippocampal neurons. J Neurosci Res 1998;54:721– 33. 14. Kihara S, Shiraishi T, Nakagawa S, Toda K, Tabuchi K. Visualization of DNA double strand breaks in the gerbil hippocampal CA1 after transient ischemia. Neurosci Lett 1994;175:133– 6. 15. Kirino T. Delayed neuronal death in the gerbil hippocampus after ischemia. Brain Res 1982;239:57– 69. 16. Lowe SW, Schmitt EM, Smith SW, Osborne BA, Jacks T. p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 1993;362:847–9. 17. Maki CG, Howley PM. Ubiquitination of p53 and p21 is
18. 19.
20. 21. 22.
23.
24. 25.
differentially affected by ionizing and UV radiation. Mol Cell Biol 1997;17:355– 63. Nagata S. Apoptosis by death factor. Cell 1997;88:355– 65. Otake M, Schull WJ. Radiation-related brain damage and growth retardation among the prenatally exposed atomic bomb survivors. Int J Radiat Biol 1998; 74:159 –71. Sakhi S, Gilmore W, Tran ND, Schreiber SS. p53deficient mice are protected against adrenalectomyinduced apoptosis. Neuroreport 1996;8:233–5. Savitz SI, Rosenbaum DM. Apoptosis in neurological disease. Neurosurgery 1998;42:555–74. Sloviter RS, Sollas AL, Dean E, Neubort S. Adrenalectomy-induced granule cell degeneration in the rat hippocampal dentate gyrus: characterization of an in vivo model of controlled neuronal death. J Comp Neurol 1993;330:324 –36. Sloviter RS, Sollas AL, Neubort S. Hippocampal dentate granule cell degeneration after adrenalectomy in the rat is not reversed by dexamethasone. Brain Res 1995;682:227–30. Suciu D. Morphometric study of the interphase nucleus in some radiosensitive and radioresistant mammalian cells. J Theor Biol 1985;113:599 – 609. Zhang H, Wheeler KT. Radiation-induced DNA damage in tumors and normal tissues. Radiat Res 1994; 140:321– 6.
COMMENTARY
Because radiation treatment has become a standard procedure in the management of brain tumors, the issue addressed in this paper is of high clinical relevance. For the first time, the authors have shown significant vulnerability of dentate granule cells in the infant mouse after exposure to a single dose of 18 Gy. A few hours after irradiation, apoptosis was found in the dentate granule cells, but not in other areas of the brain. This is an important contribution to our understanding of the well-known neuropsychological deficits caused by fractionated whole-brain radiation therapy, although single-dose irradiation might have different molecular biological effects and the conditions in humans may be different from those in rodents. Despite these restrictions, the reported results could have considerable impact on the therapeutic strategies in neuro-oncology, stressing the importance of focal irradiation (conformal stereotactic RT, brachytherapy, radiosurgery), which has a much higher potential for sparing the sensitive hippocampal structures than conventional radiation methods. Prof. Dr. med. Volker Sturm Department of Stereotactic and Functional Neurosurgery University of Cologne Cologne, Germany
Radiation-Induced Hippocampal Apoptosis
Surg Neurol 507 2000;53:503–07
This study assesses a possible mechanism of mental retardation: whole-brain radiation for brain tumors in infants and children. The dose given of 18 Gy at one shot to infantile murine whole cerebral seems to be very high compared to most clinical cases. It has already been noted that high-dose radiation induces apoptosis of cells, but vulnerability of neuronal cells to radiation in different areas of the brain has not been
clarified. Therefore, this work, which demonstrates the difference, is a valuable resource and may help prevent intellectual disturbances in children who receive radiotherapy. Kintomo Takakura, M.D., Ph.D. Department of Neurosurgery Tokyo Women’s Medical University Tokyo, Japan
orrie was able to joke about his body now. The closer he got to the end, the more he saw it as a mere shell, a container of the soul. It was withering to useless skin and bones anyhow, which made it easier to let go.
M
—Mitch Albom, “Tuesdays with Morrie”