- Email: [email protected]
Physiopathology of radiation-induced neurotoxicity
revue neurologique 167 (2011) 746–750
International meeting of the French Society of Neurology 2011
Physiopathology of radiation-induced neurotoxicity Pathophysiologie de la neurotoxicite´ radio-induite J.R. Fike Brain and Spinal Injury Center, San Francisco General Hospital, Building 1 Room 101, 1001 Potrero avenue, CA 94110 San Francisco, United States
info article
abstract
Article history:
Ionizing irradiation for the treatment of malignant brain tumors has associated with it a risk
Received 11 July 2011
of inducing serious morphologic and functional deficits. While obvious tissue damage
Accepted 26 July 2011
generally occurs after relatively high radiation doses, cognitive impairment can be seen
Published on line 1 September 2011
after lower exposures. The mechanisms responsible for cognitive injury are not well understood, but may involve neurogenesis, a process that is affected by microenvironmen-
Keywords :
tal factors including oxidative stress and inflammation. In addition, damage to neurons,
Radiation
either directly or through environmental influences may have a profound impact on
Brain
cognition. The relationships between cellular response, environmental factors and behavior
Neurogenesis
are complex and difficult to study. However, understanding such issues should provide
Oxidative stress
critical information relevant to the development of strategies and approaches to ameliorate
Neuron
or treat radiation-induced injuries that are associated with behavioral performance. # 2011 Elsevier Masson SAS. All rights reserved.
Mots cle´s : Radiothe´rapie Cerveau Neurogene`se Stress oxydatif Neurones
r e´ s u m e´ Les radiations ionisantes pour le traitement des tumeurs ce´re´brales exposent au risque de se´rieuses complications morphologiques et fonctionnelles radio-induites. Alors que les le´sions tissulaires s’observent en ge´ne´ral apre`s des doses relativement e´leve´es de radiations, des troubles cognitifs peuvent survenir apre`s une exposition plus mode´re´e. Les me´canismes responsables ne sont pas encore bien compris mais concernent la neurogene`se, un processus qui est sensible a` des facteurs microenvironnementaux comme le stress oxydatif et l’inflammation. De plus, les atteintes neuronales, directes ou au travers de facteurs environnementaux peuvent avoir un profond impact sur la cognition. Les relations entre re´ponse cellulaire, les facteurs environnementaux et comportements sont complexes et difficiles a` e´tudier. Cependant, leur compre´hension devrait fournir des informations capitales pour le de´veloppement de strate´gies visant a` ame´liorer ou traiter les le´sions radioinduites. # 2011 Elsevier Masson SAS. Tous droits re´serve´s.
E-mail address: [email protected]. 0035-3787/$ – see front matter # 2011 Elsevier Masson SAS. Tous droits re´serve´s. doi:10.1016/j.neurol.2011.07.005
747
revue neurologique 167 (2011) 746–750
1.
Introduction
After therapeutic brain irradiation, overt tissue injury generally occurs only after relatively high radiation doses (> 60 Gy, fractionated) (Tofilon and Fike, 2000). Less severe morphologic changes can occur after relatively lower doses, resulting in variable degrees of cognitive impairment, particularly in children (Butler et al., 2006; Meyers and Brown, 2006; Roman and Sperduto, 1995). Such impairment has a diverse character, but often includes hippocampus-dependent functions involving learning, memory and spatial information processing (Abayomi, 1996; Raber et al., 2004). It is particularly noteworthy that the hippocampus is an active site of neurogenesis, having multipotent stem/precursors that produce cells that migrate away and differentiate into neurons or glia (Gage, 2000). Given that there are no successful long-term treatments or preventive strategies for radiation-induced cognitive impairments, a better understanding of how cognitive injury develops after irradiation is critical for the development of approaches to manage this potentially serious complication. The cellular and molecular mechanisms underlying radiation-induced cognitive impairments are still not known, but an association between reduced neurogenesis and a variety of cognitive impairments, suggests a mechanistic link (Monje et al., 2007; Raber et al., 2004; Rola et al., 2004; Winocur et al., 2006). Furthermore, cognitive injury due to irradiation almost certainly will involve changes associated with neuronal function, either through direct cell damage or damage mediated through factors from the irradiated microenvironment such as inflammation (Fike et al., 2007; Fike et al., 2009) or oxidative stress (Fishman et al., 2009; Raber et al., 2011; Rola et al., 2007).
2.
Results and discussion
Hippocampal neurogenesis occurs in the dentate subgranular zone (SGZ), and a number of experimental reports have shown that neural stem/precursor cells in the SGZ are extremely radiosensitive (Mizumatsu et al., 2003; Monje et al., 2002; Rola et al., 2004). Additionally, the production and survival of newly born neurons is affected in a dose-dependent fashion (Fig. 1) (Mizumatsu et al., 2003). Taken together, these data, along with the studies associating altered neurogenesis with behavioral performance (Raber et al., 2004; Raber et al., 2011; Rola et al., 2004), suggest that neurogenesis may play a contributory if not causal role in the effects of irradiation on cognitive function. Considerable data exist showing that radiation effects on neurogenesis involve alterations in the microenvironment, such as oxidative stress and inflammation (Fike et al., 2007; Fike et al., 2009; Fishman et al., 2009; Monje et al., 2003; Rola et al., 2007). Altered redox state is critical in regulating the response of the CNS after a variety of insults including ionizing irradiation, and may involve increased production of reactive oxygen species (ROS), which can contribute to the spread and ultimate expression of tissue injury (Tofilon and Fike, 2000). ROS may constitute a critical environmental cue to control precursor cell survival and differentiation (Thiels et al., 2000).
% Doubled Labeled Cells
[(Fig._1)TD$IG] 80 60 40 20 0 0
2
5
10
Radiation Dose (Gy) Fig. 1 – Ionizing irradiation reduces the numbers of newly born neurons in the dentate subgranular zone. Groups of mice (n = 4) received single doses of 2–10 Gy to the head only and 1 month later received multiple injections of 5bromo-20 -deoxyurine (BrdUrd) to label cells in the S-phase of the cell cycle. One month later, tissues were collected and prepared for immunohistochemical analyses of BrdUrd-positive cells that were co-labeled with a mature neuronal marker, NeuN. Double-labeled cells were detected by confocal microscopy. After irradiation there was a significant dose-dependent decrease in the percentage of double-labeled cells (P < 0.001). Data shown were modified from: Mizumatsu et al., 2003. Les radiations ionisantes re´duisent le nombre de nouveaux neurones ne´s dans la zone sous-granulaire du gyrus dente´ de l’hippocampe. Le groupe de souris (n = 4) a rec¸u des doses uniques de 2–10 Gy uniquement a` la teˆte et un mois plus tard des injections multiples de 5-bromo-20 -deoxyurine (BrdUrd) pour marquer les cellules en phase S au cours du cycle cellulaire. Un mois plus tard, les tissus furent collecte´s et pre´pare´s pour une analyse immuno-histochimique au BrDUrd avec co-marquage avec des marqueurs neuronaux matures NeuN. Les doubles marquages furent de´tecte´s par microscopie confocale. Apre`s irradiation il y avait une diminution significative dose-de´pendante du pourcentage de cellules double-marque´es ( p < 0,001). Les images pre´sente´es ont e´te´ modifie´es d’apre`s Mizumatsu et al., 2003.
There are several pathways that mitigate the physiological and pathological effects of ROS in mammalian cells, and one of them involves the antioxidant enzyme superoxide dismutase (SOD), which exists as three genetically and geographically distinct isoenzymes (Huang et al., 1999). The SODs convert superoxide anions to hydrogen peroxide, which is then enzymatically removed by catalase and glutathione peroxidase. Although, the physiological roles of the SOD isoforms in mammalian cells are not completely understood, the extracellular isoform (EC-SOD, SOD3) is associated with certain cognitive functions, and its removal interferes with signaling cascades critical for learning (Thiels et al., 2000). Laboratory studies have been performed assessing the radiation response of animals deficient in the various SOD
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revue neurologique 167 (2011) 746–750
isoforms to determine if a persistent disruption of the redox environment affects the way irradiation impacts neurogenesis. Surprisingly, in situations of SOD1 (copper-zinc SOD), SOD2 (manganese SOD) or SOD3 deficiency, there were paradoxical effects (Fishman et al., 2009; Rola et al., 2007). In SOD knock out (KO) mice, the extent of neurogenesis prior to irradiation was decreased relative to wild type (WT) animals in all three isoforms (Fig. 2), suggesting that elevated oxidative stress was deleterious to the production and/or survival of newly born neurons. After irradiation, however, when there was a substantial and significant reduction in neurogenesis in WT mice, there were no apparent effects in animals deficient in SOD (Fig. 2) (Fishman et al., 2009; Rola et al., 2007). Additionally, in SOD3 KO mice, after irradiation, behavioral performance was improved (Raber et al., 2011). The mechanism(s) responsible for these effects is not clear, but apparently does not involve simple compensatory changes in the expression or activities of other antioxidant enzymes (Fishman et al., 2009; Rola et al., 2007). Rather it leads to a ‘‘protective’’ type of effect not unlike the adaptive (Yu and Chung, 2006) responses observed by others. One possible explanation for this finding may involve inflammation, as defined by changes in the numbers of intrinsic inflammatory cells of the brain, the microglia. After irradiation of SOD2 and SOD3 KO mice, the total number of newly born activated microglia in and around the dentate gyrus was higher that seen in WT mice (Fishman et al., 2009; Rola et al., 2007); however this was not seen in SOD1 KO mice (Fishman et al., 2009). These data are surprising after taking into account a number of recent studies suggesting that increased neuroinflammation is generally linked with an inhibition of hippocampal neurogenesis (Fike et al., 2007; Fike et al., 2009; Monje et al., 2003). Given recent data showing that microglial phenotype critically influences the ability of those cells to support or impair renewal processes (Schwartz et al., 2006), it may be that in a microenvironment characterized by persistent oxidative stress (e.g. SOD deficiency), the subsequent activation of microglia may in fact have a beneficial effect, at least in terms of neurogenesis. This would suggest that microglial response to a given stimulus (e.g. irradiation) might be context-dependent; this idea has been recently reviewed (Schwartz et al., 2006). While many questions remain, these types of data highlight the complexities associated with understanding stem/precursor cell radiation response in vivo. While there is an apparent relationship between altered neurogenesis and the development of cognitive dysfunctions after irradiation (Raber et al., 2004; Raber et al., 2011; Rola et al., 2004), simple measures of the numbers of newly born cells provide no information about their functional integration into the hippocampal circuitry or the functional integrity of mature granule cell neurons with respect to behavioral performance. One issue that has not had much emphasis to date is the idea of radiation-induced alterations in neuronal activity, particularly as it is coupled to macromolecular synthesis (gene expression) associated with learning and memory. Gene expression induced during learning produces proteins that alter the composition of hippocampal neuronal networks and provide a mechanism for translating synaptic plasticity into changes in synaptic
[(Fig._2)TD$IG]
Fig. 2 – The effects of SOD deficiency and irradiation on newly born neurons in the dentate subgranular zone. Groups (n = 4–5) of wild type (WT) C57BL6 mice or mutant mice with the genes for SOD1, SOD2 or SOD3 knocked out (KO), received a single doses of 5 Gy to the head only and 1 month later received multiple injections of 5-bromo-20 deoxyurine (BrdUrd) to label cells in the S-phase of the cell cycle. One month later, tissues were collected and prepared for immunohistochemical analyses of BrdUrdpositive cells that were co-labeled with a mature neuronal marker, NeuN. Double-labeled cells were detected by confocal microscopy. In unirradiated SOD mutant mice, there were significant (#, P < 0.05) reductions in the percentages of newly born neurons when compared to WT mice. After irradiation there was a highly significant (*, P = 0.008) reduction in newly born neurons in WT mice, but no reductions in any of the mutant mice. Data shown were modified from: Rola et al., 2007 and Fishman et al., 2009. Conse´quences du de´ficit en SOD et de l’irradiation sur des neurones ne´s dans la zone sous-granulaire du gyrus dente´. Les groupes (n = 4–5) de souris sauvages (WT) C57BL6 ou mute´es avec les ge`nes SOD1, SOD2 ou SOD3 knock-out (KO), recevaient une dose unique de 5 Gy a` la teˆte et un mois plus tard des injections multiples de 5-bromo-20 -deoxyurine (BrdUrd) pour marquer les cellules en phase S du cycle cellulaire. Un mois plus tard, les tissus furent collecte´s et pre´pare´s pour une analyse immuno-histochimique au BrDUrd avec co-marquage des marqueurs neuronaux matures NeuN. Les doubles marquages furent de´tecte´s par microscopie confocale. Chez les souris SOD non irradie´es, on observait une re´duction significative du pourcentage de neurones nouveaune´s compare´ aux souris sauvages (#, P < 0,05). Apre`s irradiation, on observait une re´duction significative de neurones nouveau-ne´s (*, P = 0,008) chez les souris sauvages mais pas chez les mute´es. Les images pre´sente´es ont e´te´ modifie´es d’apre`s : Rola et al., 2007 et Fishman et al., 2009.
strength (memory). Among the immediate early genes (IEGs), Arc (activity-regulated cytoskeleton-associated protein) has a distinct role in modulating hippocampal synaptic plasticity (Lyford et al., 1995), and Arc protein is essential for consolidation of synaptic plasticity and memory (Plath et al., 2006). Taken together, this information provides a
revue neurologique 167 (2011) 746–750
mechanistic link between Arc and hippocampal-dependent functions, and provides a strong rationale for using Arc expression to assess specific neuronal activities associated with cognitive impairments induced by cranial irradiation. When animals are placed in a novel learning environment, Arc mRNA is induced in neuronal nuclei within 2–5 minutes, is translocated into the cytoplasm a few minutes later, and within 30 minutes is translated into protein (Fike et al., 2009; Rosi et al., 2008). We irradiated mice with 0 Gy or 10 Gy, a dose that induces cognitive deficits (Raber et al., 2004). Two months later, we used exploration of a novel environment to induce Arc. Following exploration, the number of neurons expressing Arc in the hippocampus was significantly higher than that seen in caged control mice that did not explore the novel environment. Irradiation reduced the fractions of neurons, expressing behaviorally induced Arc mRNA and Arc protein in the DG (Table 1), without any radiation-induced cell loss (Rosi et al., 2008). Given the information on relative cell numbers in the dentate gyrus (Kempermann et al., 1997), the modest changes in Arc mRNA and Arc protein we saw in our study represent a relatively larger number of cells in the dentate gyrus and could be extremely important for proper hippocampal function. These data suggest that addressing the molecular distribution of Arc at the level of mRNA and protein could provide a novel way to consider radiation-induced brain injury. Additionally, it has recently been shown that the proportion of newly born neurons that express Arc in response to a learning experience is nearly two fold higher than what is seen in mature granule cell neurons (Ramirez-Amaya et al., 2006), and those cells were preferentially recruited into circuits supporting spatial memory (Kee et al., 2007). This provides a potential mechanistic link between Arc expression, neurogenesis and behavior, and may offer new insight into how irradiation may impact cognitive function. While there is still considerable uncertainty regarding how ionizing irradiation affects cognition, data are becoming available that allow us to consider new ideas about how to
Table 1 – The effects of irradiation on the fractions of neurons expressing Arc mRNA and Arc protein in mice allowed to explore a novel environment. One week after irradiation there was no apparent effect of irradiation on Arc mRNA and a reduction in Arc protein (*, P < 0.05). Two months after irradiation there were significant reductions in both Arc mRNA and Arc protein. Values shown represent the mean of six mice; error bars represent standard errors of the mean. L’irradiation de la fraction de neurones exprimant Arc ARNm et la prote´ine ARC permirent d’explorer un nouvel environnement. Une semaine apre`s l’irradiation, on n’observait pas d’effet apparent sur Arc ARNm et une re´duction de la prote´ine ARC (*, p < 0,05). Deux mois apre`s l’irradiation, on observait une re´duction significative de Arc ARNm et de la prote´ine ARC. Les valeurs pre´sente´es correspondent a` la moyenne de six souris. One Week
Arc mRNA Arc Protein
Two months
0 Gy
10 Gy
0 Gy
10 Gy
6.93 0.61 6.63 0.43
6.67 0.67 5.15 0.23*
6.39 0.72 5.99 0.57
3.48 0.76* 3.58 0.76*
749
view this serious complication of cranial radiotherapy. These ideas include neural stem/precursor cells response and how they are impacted by microenvironmental factors such as oxidative stress and inflammation. Furthermore, the ability to assess neuronal function will provide a means to address cells that survive irradiation but whose function may be affected. Understanding such issues should provide critical information relevant to the development of strategies and approaches to ameliorate or treat radiation-induced injuries that are associated with behavioral performance.
Disclosure of interest The author declares that he has no conflicts of interest concerning this article.
Acknowledgements Much of the work reviewed here was supported by NIH-grant R01 NS46051.
references
Abayomi OK. Pathogenesis of irradiation-induced cognitive dysfunction. Acta Oncol 1996;35:659–63. Butler JM, Rapp SR, Shaw EG. Managing the cognitive effects of brain tumor radiation therapy. Curr Treat Options Oncol 2006;7:517–23. Fike JR, Rola R, Limoli CL. Radiation response of neural precursor cells. Neurosurg Clin N Am 2007;18:115–27. Fike JR, Rosi S, Limoli CL. Neural precursor cells and central nervous system radiation sensitivity. Semin Radiat Oncol 2009;19:122–32. Fishman K, Baure J, Zou Y, Huang TT, Andres-Mach M, Rola R, et al. Radiation-induced reductions in neurogenesis are ameliorated in mice deficient in CuZnSOD or MnSOD. Free Radic Biol Med 2009;47:1459–67. Gage FH. Mammalian neural stem cells. Science 2000;287: 1433–8. Huang TT, Carlson EJ, Raineri I, Gillespie AM, Kozy H, Epstein CJ. The use of transgenic and mutant mice to study oxygen free radical metabolism. Ann N Y Acad Sci 1999;893:95–112. Kee N, Teixeira CM, Wang AH, Frankland PW. Preferential incorporation of adult-generated granule cells into spatial memory networks in the dentate gyrus. Nat Neurosci 2007;10:355–62. Kempermann G, Kuhn HG, Gage FH. Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proc Natl Acad Sci U S A 1997;94:10409–14. Lyford GL, Yamagata K, Kaufmann WE, et al. Arc, a growth factor and activity-regulated gene, encodes a novel cytoskeleton-associated protein that is enriched in neuronal dendrites. Neuron 1995;14:433–45. Meyers CA, Brown PD. Role and relevance of neurocognitive assessment in clinical trials of patients with CNS tumors. J Clin Oncol 2006;24:1305–9. Mizumatsu S, Monje ML, Morhardt DR, Rola R, Palmer TD, Fike JR. Extreme sensitivity of adult neurogenesis to low doses of x-irradiation. Cancer Res 2003;63:4021–7. Monje ML, Mizumatsu S, Fike JR, Palmer TD. Irradiation induces neural precursor-cell dysfunction. Nat Med 2002;8:955–62.
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Monje ML, Toda H, Palmer TD. Inflammatory blockade restores adult hippocampal neurogenesis. Science 2003; 302:1760–5. Monje ML, Vogel H, Masek M, Ligon KL, Fisher PG, Palmer TD. Impaired human hippocampal neurogenesis after treatment for central nervous system malignancies. Ann Neurol 2007;62:515–20. Plath N, Ohana O, Dammermann B, Errington ML, Schmitz D, Gross C, et al. Arc/Arg 3.1 is essential for the consolidation of synaptic plasticity and memories. Neuron 2006;52:437–44. Raber J, Rola R, LeFevour A, Morhardt D, Curley J, Mizumatsu S, et al. Radiation-induced cognitive impairments are associated with changes in indicators of hippocampal neurogenesis. Radiat Res 2004;162:39–47. Raber J, Villasana L, Rosenberg J, Zou Y, Huang TT, Fike JR. Irradiation enhances hippocampus-dependent cognition in mice deficient in extracellular superoxide dismutase. Hippocampus 2011;21:72–80. Ramirez-Amaya V, Marrone DF, Gage FH, Worley PF, Barnes CA. Integration of new neurons into functional neural networks. J Neurosci 2006;26:12237–41. Rola R, Raber J, Rizk A, Otsuka S, VandenBerg SR, Morhardt DR, et al. Radiation-induced impairment of hippocampal neurogenesis is associated with cognitive deficits in young mice. Exp Neurol 2004;188:316–30.
Rola R, Zou Y, Huang TT, Fishman K, Baure J, Rosi S, et al. Lack of EC-SOD in the microenvironment impacts radiation-induced changes in neurogenesis. Free Radic Biol Med 2007;42:1133–45. Roman DD, Sperduto PW. Neuropsychological effects of cranial radiation: current knowledge and future directions. Int J Radiat Oncol Biol Phys 1995;31:983–98. Rosi S, Andres-Mach M, Fishman KM, Levy W, Ferguson RA, Fike JR. Cranial irradiation alters the behaviorally induced immediate-early gene arc (activity-regulated cytoskeletonassociated protein). Cancer Res 2008;68:9763–70. Schwartz M, Butovsky O, Bruck W, Hanisch UK. Microglial phenotype: is the commitment reversible? Trends Neurosci 2006;29:68–74. Thiels E, Urban NN, Gonzalez-Burgos GR, Kanterewicz BI, Barrionuevo G, Chu CT, et al. Impairment of long-term potentiation and associative memory in mice that over express extracellular superoxide dismutase. J Neurosci 2000;20:7631–9. Tofilon PJ, Fike JR. The radio response of the central nervous system: a dynamic process. Radiat Res 2000;153:357–70. Winocur G, Wojtowicz JM, Sekeres M, Snyder JS, Wang S. Inhibition of neurogenesis interferes with hippocampusdependent memory function. Hippocampus 2006;16:296–304. Yu BP, Chung HY. Adaptive mechanisms to oxidative stress during aging. Mech Ageing Dev 2006;127:436–43.
International meeting of the French Society of Neurology 2011
Physiopathology of radiation-induced neurotoxicity Pathophysiologie de la neurotoxicite´ radio-induite J.R. Fike Brain and Spinal Injury Center, San Francisco General Hospital, Building 1 Room 101, 1001 Potrero avenue, CA 94110 San Francisco, United States
info article
abstract
Article history:
Ionizing irradiation for the treatment of malignant brain tumors has associated with it a risk
Received 11 July 2011
of inducing serious morphologic and functional deficits. While obvious tissue damage
Accepted 26 July 2011
generally occurs after relatively high radiation doses, cognitive impairment can be seen
Published on line 1 September 2011
after lower exposures. The mechanisms responsible for cognitive injury are not well understood, but may involve neurogenesis, a process that is affected by microenvironmen-
Keywords :
tal factors including oxidative stress and inflammation. In addition, damage to neurons,
Radiation
either directly or through environmental influences may have a profound impact on
Brain
cognition. The relationships between cellular response, environmental factors and behavior
Neurogenesis
are complex and difficult to study. However, understanding such issues should provide
Oxidative stress
critical information relevant to the development of strategies and approaches to ameliorate
Neuron
or treat radiation-induced injuries that are associated with behavioral performance. # 2011 Elsevier Masson SAS. All rights reserved.
Mots cle´s : Radiothe´rapie Cerveau Neurogene`se Stress oxydatif Neurones
r e´ s u m e´ Les radiations ionisantes pour le traitement des tumeurs ce´re´brales exposent au risque de se´rieuses complications morphologiques et fonctionnelles radio-induites. Alors que les le´sions tissulaires s’observent en ge´ne´ral apre`s des doses relativement e´leve´es de radiations, des troubles cognitifs peuvent survenir apre`s une exposition plus mode´re´e. Les me´canismes responsables ne sont pas encore bien compris mais concernent la neurogene`se, un processus qui est sensible a` des facteurs microenvironnementaux comme le stress oxydatif et l’inflammation. De plus, les atteintes neuronales, directes ou au travers de facteurs environnementaux peuvent avoir un profond impact sur la cognition. Les relations entre re´ponse cellulaire, les facteurs environnementaux et comportements sont complexes et difficiles a` e´tudier. Cependant, leur compre´hension devrait fournir des informations capitales pour le de´veloppement de strate´gies visant a` ame´liorer ou traiter les le´sions radioinduites. # 2011 Elsevier Masson SAS. Tous droits re´serve´s.
E-mail address: [email protected]. 0035-3787/$ – see front matter # 2011 Elsevier Masson SAS. Tous droits re´serve´s. doi:10.1016/j.neurol.2011.07.005
747
revue neurologique 167 (2011) 746–750
1.
Introduction
After therapeutic brain irradiation, overt tissue injury generally occurs only after relatively high radiation doses (> 60 Gy, fractionated) (Tofilon and Fike, 2000). Less severe morphologic changes can occur after relatively lower doses, resulting in variable degrees of cognitive impairment, particularly in children (Butler et al., 2006; Meyers and Brown, 2006; Roman and Sperduto, 1995). Such impairment has a diverse character, but often includes hippocampus-dependent functions involving learning, memory and spatial information processing (Abayomi, 1996; Raber et al., 2004). It is particularly noteworthy that the hippocampus is an active site of neurogenesis, having multipotent stem/precursors that produce cells that migrate away and differentiate into neurons or glia (Gage, 2000). Given that there are no successful long-term treatments or preventive strategies for radiation-induced cognitive impairments, a better understanding of how cognitive injury develops after irradiation is critical for the development of approaches to manage this potentially serious complication. The cellular and molecular mechanisms underlying radiation-induced cognitive impairments are still not known, but an association between reduced neurogenesis and a variety of cognitive impairments, suggests a mechanistic link (Monje et al., 2007; Raber et al., 2004; Rola et al., 2004; Winocur et al., 2006). Furthermore, cognitive injury due to irradiation almost certainly will involve changes associated with neuronal function, either through direct cell damage or damage mediated through factors from the irradiated microenvironment such as inflammation (Fike et al., 2007; Fike et al., 2009) or oxidative stress (Fishman et al., 2009; Raber et al., 2011; Rola et al., 2007).
2.
Results and discussion
Hippocampal neurogenesis occurs in the dentate subgranular zone (SGZ), and a number of experimental reports have shown that neural stem/precursor cells in the SGZ are extremely radiosensitive (Mizumatsu et al., 2003; Monje et al., 2002; Rola et al., 2004). Additionally, the production and survival of newly born neurons is affected in a dose-dependent fashion (Fig. 1) (Mizumatsu et al., 2003). Taken together, these data, along with the studies associating altered neurogenesis with behavioral performance (Raber et al., 2004; Raber et al., 2011; Rola et al., 2004), suggest that neurogenesis may play a contributory if not causal role in the effects of irradiation on cognitive function. Considerable data exist showing that radiation effects on neurogenesis involve alterations in the microenvironment, such as oxidative stress and inflammation (Fike et al., 2007; Fike et al., 2009; Fishman et al., 2009; Monje et al., 2003; Rola et al., 2007). Altered redox state is critical in regulating the response of the CNS after a variety of insults including ionizing irradiation, and may involve increased production of reactive oxygen species (ROS), which can contribute to the spread and ultimate expression of tissue injury (Tofilon and Fike, 2000). ROS may constitute a critical environmental cue to control precursor cell survival and differentiation (Thiels et al., 2000).
% Doubled Labeled Cells
[(Fig._1)TD$IG] 80 60 40 20 0 0
2
5
10
Radiation Dose (Gy) Fig. 1 – Ionizing irradiation reduces the numbers of newly born neurons in the dentate subgranular zone. Groups of mice (n = 4) received single doses of 2–10 Gy to the head only and 1 month later received multiple injections of 5bromo-20 -deoxyurine (BrdUrd) to label cells in the S-phase of the cell cycle. One month later, tissues were collected and prepared for immunohistochemical analyses of BrdUrd-positive cells that were co-labeled with a mature neuronal marker, NeuN. Double-labeled cells were detected by confocal microscopy. After irradiation there was a significant dose-dependent decrease in the percentage of double-labeled cells (P < 0.001). Data shown were modified from: Mizumatsu et al., 2003. Les radiations ionisantes re´duisent le nombre de nouveaux neurones ne´s dans la zone sous-granulaire du gyrus dente´ de l’hippocampe. Le groupe de souris (n = 4) a rec¸u des doses uniques de 2–10 Gy uniquement a` la teˆte et un mois plus tard des injections multiples de 5-bromo-20 -deoxyurine (BrdUrd) pour marquer les cellules en phase S au cours du cycle cellulaire. Un mois plus tard, les tissus furent collecte´s et pre´pare´s pour une analyse immuno-histochimique au BrDUrd avec co-marquage avec des marqueurs neuronaux matures NeuN. Les doubles marquages furent de´tecte´s par microscopie confocale. Apre`s irradiation il y avait une diminution significative dose-de´pendante du pourcentage de cellules double-marque´es ( p < 0,001). Les images pre´sente´es ont e´te´ modifie´es d’apre`s Mizumatsu et al., 2003.
There are several pathways that mitigate the physiological and pathological effects of ROS in mammalian cells, and one of them involves the antioxidant enzyme superoxide dismutase (SOD), which exists as three genetically and geographically distinct isoenzymes (Huang et al., 1999). The SODs convert superoxide anions to hydrogen peroxide, which is then enzymatically removed by catalase and glutathione peroxidase. Although, the physiological roles of the SOD isoforms in mammalian cells are not completely understood, the extracellular isoform (EC-SOD, SOD3) is associated with certain cognitive functions, and its removal interferes with signaling cascades critical for learning (Thiels et al., 2000). Laboratory studies have been performed assessing the radiation response of animals deficient in the various SOD
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revue neurologique 167 (2011) 746–750
isoforms to determine if a persistent disruption of the redox environment affects the way irradiation impacts neurogenesis. Surprisingly, in situations of SOD1 (copper-zinc SOD), SOD2 (manganese SOD) or SOD3 deficiency, there were paradoxical effects (Fishman et al., 2009; Rola et al., 2007). In SOD knock out (KO) mice, the extent of neurogenesis prior to irradiation was decreased relative to wild type (WT) animals in all three isoforms (Fig. 2), suggesting that elevated oxidative stress was deleterious to the production and/or survival of newly born neurons. After irradiation, however, when there was a substantial and significant reduction in neurogenesis in WT mice, there were no apparent effects in animals deficient in SOD (Fig. 2) (Fishman et al., 2009; Rola et al., 2007). Additionally, in SOD3 KO mice, after irradiation, behavioral performance was improved (Raber et al., 2011). The mechanism(s) responsible for these effects is not clear, but apparently does not involve simple compensatory changes in the expression or activities of other antioxidant enzymes (Fishman et al., 2009; Rola et al., 2007). Rather it leads to a ‘‘protective’’ type of effect not unlike the adaptive (Yu and Chung, 2006) responses observed by others. One possible explanation for this finding may involve inflammation, as defined by changes in the numbers of intrinsic inflammatory cells of the brain, the microglia. After irradiation of SOD2 and SOD3 KO mice, the total number of newly born activated microglia in and around the dentate gyrus was higher that seen in WT mice (Fishman et al., 2009; Rola et al., 2007); however this was not seen in SOD1 KO mice (Fishman et al., 2009). These data are surprising after taking into account a number of recent studies suggesting that increased neuroinflammation is generally linked with an inhibition of hippocampal neurogenesis (Fike et al., 2007; Fike et al., 2009; Monje et al., 2003). Given recent data showing that microglial phenotype critically influences the ability of those cells to support or impair renewal processes (Schwartz et al., 2006), it may be that in a microenvironment characterized by persistent oxidative stress (e.g. SOD deficiency), the subsequent activation of microglia may in fact have a beneficial effect, at least in terms of neurogenesis. This would suggest that microglial response to a given stimulus (e.g. irradiation) might be context-dependent; this idea has been recently reviewed (Schwartz et al., 2006). While many questions remain, these types of data highlight the complexities associated with understanding stem/precursor cell radiation response in vivo. While there is an apparent relationship between altered neurogenesis and the development of cognitive dysfunctions after irradiation (Raber et al., 2004; Raber et al., 2011; Rola et al., 2004), simple measures of the numbers of newly born cells provide no information about their functional integration into the hippocampal circuitry or the functional integrity of mature granule cell neurons with respect to behavioral performance. One issue that has not had much emphasis to date is the idea of radiation-induced alterations in neuronal activity, particularly as it is coupled to macromolecular synthesis (gene expression) associated with learning and memory. Gene expression induced during learning produces proteins that alter the composition of hippocampal neuronal networks and provide a mechanism for translating synaptic plasticity into changes in synaptic
[(Fig._2)TD$IG]
Fig. 2 – The effects of SOD deficiency and irradiation on newly born neurons in the dentate subgranular zone. Groups (n = 4–5) of wild type (WT) C57BL6 mice or mutant mice with the genes for SOD1, SOD2 or SOD3 knocked out (KO), received a single doses of 5 Gy to the head only and 1 month later received multiple injections of 5-bromo-20 deoxyurine (BrdUrd) to label cells in the S-phase of the cell cycle. One month later, tissues were collected and prepared for immunohistochemical analyses of BrdUrdpositive cells that were co-labeled with a mature neuronal marker, NeuN. Double-labeled cells were detected by confocal microscopy. In unirradiated SOD mutant mice, there were significant (#, P < 0.05) reductions in the percentages of newly born neurons when compared to WT mice. After irradiation there was a highly significant (*, P = 0.008) reduction in newly born neurons in WT mice, but no reductions in any of the mutant mice. Data shown were modified from: Rola et al., 2007 and Fishman et al., 2009. Conse´quences du de´ficit en SOD et de l’irradiation sur des neurones ne´s dans la zone sous-granulaire du gyrus dente´. Les groupes (n = 4–5) de souris sauvages (WT) C57BL6 ou mute´es avec les ge`nes SOD1, SOD2 ou SOD3 knock-out (KO), recevaient une dose unique de 5 Gy a` la teˆte et un mois plus tard des injections multiples de 5-bromo-20 -deoxyurine (BrdUrd) pour marquer les cellules en phase S du cycle cellulaire. Un mois plus tard, les tissus furent collecte´s et pre´pare´s pour une analyse immuno-histochimique au BrDUrd avec co-marquage des marqueurs neuronaux matures NeuN. Les doubles marquages furent de´tecte´s par microscopie confocale. Chez les souris SOD non irradie´es, on observait une re´duction significative du pourcentage de neurones nouveaune´s compare´ aux souris sauvages (#, P < 0,05). Apre`s irradiation, on observait une re´duction significative de neurones nouveau-ne´s (*, P = 0,008) chez les souris sauvages mais pas chez les mute´es. Les images pre´sente´es ont e´te´ modifie´es d’apre`s : Rola et al., 2007 et Fishman et al., 2009.
strength (memory). Among the immediate early genes (IEGs), Arc (activity-regulated cytoskeleton-associated protein) has a distinct role in modulating hippocampal synaptic plasticity (Lyford et al., 1995), and Arc protein is essential for consolidation of synaptic plasticity and memory (Plath et al., 2006). Taken together, this information provides a
revue neurologique 167 (2011) 746–750
mechanistic link between Arc and hippocampal-dependent functions, and provides a strong rationale for using Arc expression to assess specific neuronal activities associated with cognitive impairments induced by cranial irradiation. When animals are placed in a novel learning environment, Arc mRNA is induced in neuronal nuclei within 2–5 minutes, is translocated into the cytoplasm a few minutes later, and within 30 minutes is translated into protein (Fike et al., 2009; Rosi et al., 2008). We irradiated mice with 0 Gy or 10 Gy, a dose that induces cognitive deficits (Raber et al., 2004). Two months later, we used exploration of a novel environment to induce Arc. Following exploration, the number of neurons expressing Arc in the hippocampus was significantly higher than that seen in caged control mice that did not explore the novel environment. Irradiation reduced the fractions of neurons, expressing behaviorally induced Arc mRNA and Arc protein in the DG (Table 1), without any radiation-induced cell loss (Rosi et al., 2008). Given the information on relative cell numbers in the dentate gyrus (Kempermann et al., 1997), the modest changes in Arc mRNA and Arc protein we saw in our study represent a relatively larger number of cells in the dentate gyrus and could be extremely important for proper hippocampal function. These data suggest that addressing the molecular distribution of Arc at the level of mRNA and protein could provide a novel way to consider radiation-induced brain injury. Additionally, it has recently been shown that the proportion of newly born neurons that express Arc in response to a learning experience is nearly two fold higher than what is seen in mature granule cell neurons (Ramirez-Amaya et al., 2006), and those cells were preferentially recruited into circuits supporting spatial memory (Kee et al., 2007). This provides a potential mechanistic link between Arc expression, neurogenesis and behavior, and may offer new insight into how irradiation may impact cognitive function. While there is still considerable uncertainty regarding how ionizing irradiation affects cognition, data are becoming available that allow us to consider new ideas about how to
Table 1 – The effects of irradiation on the fractions of neurons expressing Arc mRNA and Arc protein in mice allowed to explore a novel environment. One week after irradiation there was no apparent effect of irradiation on Arc mRNA and a reduction in Arc protein (*, P < 0.05). Two months after irradiation there were significant reductions in both Arc mRNA and Arc protein. Values shown represent the mean of six mice; error bars represent standard errors of the mean. L’irradiation de la fraction de neurones exprimant Arc ARNm et la prote´ine ARC permirent d’explorer un nouvel environnement. Une semaine apre`s l’irradiation, on n’observait pas d’effet apparent sur Arc ARNm et une re´duction de la prote´ine ARC (*, p < 0,05). Deux mois apre`s l’irradiation, on observait une re´duction significative de Arc ARNm et de la prote´ine ARC. Les valeurs pre´sente´es correspondent a` la moyenne de six souris. One Week
Arc mRNA Arc Protein
Two months
0 Gy
10 Gy
0 Gy
10 Gy
6.93 0.61 6.63 0.43
6.67 0.67 5.15 0.23*
6.39 0.72 5.99 0.57
3.48 0.76* 3.58 0.76*
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view this serious complication of cranial radiotherapy. These ideas include neural stem/precursor cells response and how they are impacted by microenvironmental factors such as oxidative stress and inflammation. Furthermore, the ability to assess neuronal function will provide a means to address cells that survive irradiation but whose function may be affected. Understanding such issues should provide critical information relevant to the development of strategies and approaches to ameliorate or treat radiation-induced injuries that are associated with behavioral performance.
Disclosure of interest The author declares that he has no conflicts of interest concerning this article.
Acknowledgements Much of the work reviewed here was supported by NIH-grant R01 NS46051.
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