Marie Kalm

Transient Inflammation in Neurogenic Regions after Irradiation of the Developing Brain

Abstract Kalm, M., Fukuda, A., Fukuda, H., Öhrfelt, A., Lannering, B., Björk-Eriksson, T., Blennow, K., Márky, I. and Blomgren, K. Transient Inflammation in Neurogenic Regions after Irradiation of the Developing Brain. Radiat. Res. 171, 66–76 (2009). We characterized the inflammatory response after a single dose of 8 Gy to the brains of postnatal day 9 rats. Affymetrix gene chips revealed activation of multiple inflammatory mechanisms in the acute phase, 6 h after irradiation. In the subacute phase, 7 days after irradiation, genes related to neurogenesis and cell cycle were down-regulated, but glial fibrillary acidic protein (GFAP) was up-regulated. The concentrations of 14 different cytokines and chemokines were measured using a microsphere-based xMAP™ technology. CCL2, Gro/KC and IL-1α were the most strongly up-regulated 6 h after irradiation. CCL2 was expressed in astrocytes and microglia in the dentate gyrus and the subventricular zone (SVZ). Hypertrophy, but not hyperplasia, of astrocytes was demonstrated 7 days after irradiation. In summary, we found transient activation of multiple inflammatory mechanisms in the acute phase (6 h) after irradiation and activation of astrocytes in the subacute phase (7 days) after irradiation. It remains to be elucidated whether these transient changes are involved in the persistent effects of radiation observed on neurogenesis and cognition in rodents.

Sex-dependent differences in behavior and hippocampal neurogenesis after irradiation to the young mouse brain.

Cranial radiotherapy in the treatment of pediatric malignancies may lead to cognitive deficits, and girls suffer more severe deficits than boys. However, most experimental studies are performed on male animals only. Our aim was to investigate possible long‐term gender differences in response to cranial irradiation (IR). Basal neurogenesis in non‐irradiated mice was higher in females but this was not apparent until the animals were adult. Male and female C57BL/6J mice received a single dose of 8 Gy to the whole brain on postnatal day 14 and were killed 6 h or 4 months later. Proliferation in the subgranular zone of the dentate gyrus in the hippocampus, as judged by the number of phosphohistone H3‐positive cells, was reduced by half 6 h after IR in both males and females. The reduced proliferation was still obvious 4 months after IR. Consequently, the continuous addition of new neurons to the granule cell layer (GCL) during brain growth was reduced in irradiated mice, and the reduction was more pronounced in females. This resulted in hampered growth of the GCL, reduced bromodeoxyuridine incorporation in adulthood, and severely reduced adult neurogenesis, as judged by the number of doublecortin‐positive cells in the GCL. In an open‐field test, locomotor activity was increased in both males and females after IR and anxiety levels were increased, more so in females. In an IntelliCage test, place learning was impaired by IR in females but not males.

Loss of hippocampal neurogenesis, increased novelty-induced activity, decreased home cage activity, and impaired reversal learning one year after irradiation of the young mouse brain.

Radiotherapy is a major cause of long-term complications in survivors of pediatric brain tumors. These complications include intellectual and memory impairments as well as perturbed growth and puberty. We investigated the long-term effects of a single 8 Gy irradiation dose to the brains of 14-day-old mice. Behavior was assessed one year after irradiation using IntelliCage and open field, followed by immunohistochemical investigation of proliferation and neurogenesis in the dentate gyrus of the hippocampus. We found a 61% reduction in proliferation and survival (BrdU incorporation 4 weeks prior to sacrifice), 99% decrease in neurogenesis (number of doublecortin-positive cells) and gliosis (12% higher astrocyte density) one year following irradiation. Irradiated animals displayed increased activity in a novel environment but decreased activity in their home cage. Place learning in the IntelliCage was unaffected by irradiation but reversal learning was impaired. Irradiated animals persevered in visiting previously correct corners to a higher extent compared to control animals. Hence, despite the virtual absence of neurogenesis in these old mice, spatial learning could take place. Reversal learning however, where a previous memory was replaced with a new one, was partly impaired. This model is useful to study the so called late effects of radiotherapy to the young brain and to evaluate possible interventions.

Irradiation-induced loss of microglia in the young brain.

Irradiation-induced loss of neural stem and progenitor cells may contribute to cognitive deficits. Furthermore, subsequent inflammation inhibits neural progenitor cell differentiation. Here we have characterized the microglia response after a single dose of 8 Gy to the brains of postnatal day 9 or 21 rats. The number of Iba-1-positive microglia increased 6 h after IR but had decreased 7 days later, below control levels, and this decrease was more pronounced in P9 rats. Active caspase-3 and TUNEL staining revealed irradiation-induced microglia death. This age-dependent IR-induced loss of microglia likely affects both the response to IR and further brain development.

Irradiation to the young mouse brain caused long-term, progressive depletion of neurogenesis but did not disrupt the neurovascular niche

We investigated the effects of ionizing radiation on microvessel structure and complexity in the hippocampus. We also assessed neurogenesis and the neurovascular niche. Postnatal day 14 male C57BL/6 mice received a single dose of 8 Gy to the whole brain and were killed 6 hours, 1 week, 7 weeks, or 1 year later. Irradiation decreased the total number of microvessels and branching points from 1 week onwards and decreased the total microvessel area 1 and 7 weeks after irradiation. After an initial increase in vascular parameter densities, concomitant with reduced growth of the hippocampus, the densities normalized with time, presumably adapting to the needs of the surrounding nonvascular tissue. Irradiation decreased the number of neural stem and progenitor cells in the hippocampus. The relative loss increased with time, resulting in almost completely ablated neurogenesis (DCX+ cells) 1 year after irradiation (77% decreased 1 week, 86% decreased 7 weeks, and 98% decreased 1 year after irradiation compared with controls). After irradiation, the distance between undifferentiated stem cells and microvessels was unaffected, and very few dying endothelial cells were detected. Taken together, these results indicate that the vasculature adjusts to the surrounding neural and glial tissue after irradiation, not vice-versa.

Learning and Activity after Irradiation of the Young Mouse Brain Analyzed in Adulthood Using Unbiased Monitoring in a Home Cage Environment

Abstract Cranial radiotherapy during the treatment of pediatric malignancies may cause adverse late effects. It is important to find methods to assess the functional effects of ionizing radiation in animal models and to evaluate the possible ameliorating effects of preventive or reparative treatment strategies. We investigated the long-term effects of a single 8-Gy radiation dose to the brains of 14-day-old mice. Activity and learning were evaluated in adulthood using open field and trace fear conditioning (TFC). These established methods were compared with the novel IntelliCage platform, which enables unbiased analysis of both activity and learning over time in a home cage environment. Neither activity nor learning was changed after irradiation, as judged by the open field and TFC analyses. The IntelliCage, however, revealed both altered activity and learning impairment after irradiation. Place learning and reversal learning were both impaired in the IntelliCage 3 months after irradiation. These results indicate that activity and learning should be assessed using multiple methods and that unbiased analysis over time in a home cage environment may offer advantages in the detection of subtle radiation-induced effects on the young brain.

Altered cognitive performance and synaptic function in the hippocampus of mice lacking C3.

Previous work implicated the complement system in adult neurogenesis as well as elimination of synapses in the developing and injured CNS. In the present study, we used mice lacking the third complement component (C3) to elucidate the role the complement system plays in hippocampus-dependent learning and synaptic function. We found that the constitutive absence of C3 is associated with enhanced place and reversal learning in adult mice. Our findings of lower release probability at CA3-CA1 glutamatergic synapses in combination with unaltered overall efficacy of these synapses in C3 deficient mice implicate C3 as a negative regulator of the number of functional glutamatergic synapses in the hippocampus. The C3 deficient mice showed no signs of spontaneous epileptiform activity in the hippocampus. We conclude that C3 plays a role in the regulation of the number and function of glutamatergic synapses in the hippocampus and exerts negative effects on hippocampus-dependent cognitive performance.

Different reactions to irradiation in the juvenile and adult hippocampus

Abstract Purpose: Cranial radiotherapy is an important tool in the cure of primary brain tumors. Unfortunately, it is associated with late-appearing toxicity to the normal brain tissue, including cognitive impairment, particularly in children. The underlying mechanisms are not fully understood but involve changes in hippocampal neurogenesis. Recent studies report essentially different responses in the juvenile and the adult brain after irradiation, but this has never been verified in a comparative study. Materials and methods: We subjected juvenile (9-day-old) and adult (6-month-old) male rats to a single dose of 6 Gray (Gy) whole brain irradiation and euthanized them 6 hours, 7 days or 4 weeks later. Hippocampal lysates were analyzed for caspase-3 activity (apoptosis) and the expression of cytokines, chemokines and growth factors. Four weeks after irradiation, the number of microglia (expressing ionized calcium-binding adapter molecule 1, Iba-1), activated microglia (expressing cluster of differentiation 68 [CD68]), bromodeoxyuridine (BrdU) incorporation and granule cell layer (GCL) volume were assessed. Results: The major findings were (i) higher baseline BrdU incorporation (cell proliferation) in juvenile than in adult controls, which explains the increased susceptibility to irradiation and higher level of acute cell death (caspase activity) in juvenile rats, leading to impaired growth and subsequently a smaller dentate gyrus volume 4 weeks after irradiation, (ii) more activated (CD68-positive) microglia in adult compared to juvenile rats, regardless of irradiation, and (iii) differently expressed cytokines and chemokines after cranial irradiation in the juvenile compared to the adult rat hippocampus, indicating a more pro-inflammatory response in adult brains. Conclusion: We found essentially diverse irradiation reactions in the juvenile compared to the adult hippocampus, indicating different mechanisms involved in degeneration and regeneration after injury. Strategies to ameliorate the cognitive deficits after cranial radiotherapy should therefore likely be adapted to the developmental level of the brain.

Irradiation to the young mouse brain impaired white matter growth more in females than in males

Modern therapy cures 80% of all children with brains tumors, but may also cause long-lasting side effects, so called late effects. Radiotherapy is particularly prone to cause severe late effects, such as intellectual impairment. The extent and nature of the resulting cognitive deficits may be influenced by age, treatment and gender, where girls suffer more severe late effects than boys. The reason for this difference between boys and girls is unknown, but very few experimental studies have addressed this issue. Our aim was to investigate the effects of ionizing radiation on the corpus callosum (CC) in both male and female mice. We found that a single dose of 8 Gray (Gy) to the brains of postnatal day 14 mice induced apoptosis in the CC and reduced the number of proliferating cells by one third, as judged by the number of phospho-histone H3 positive cells 6 h after irradiation (IR). BrdU incorporation was reduced (62% and 42% lower in females and males, respectively) and the number of oligodendrocytes (Olig2+ cells) was lower (43% and 21% fewer in females and males, respectively) 4 months after IR, so the lack of developing and differentiated cells was more pronounced in females. The number of microglia was unchanged in females but increased in males at this late time point. The density of microvessel profiles was unchanged by IR. This single, moderate dose of 8 Gy impaired the brain growth to some extent (8.1% and 0.4% lower brain/body weight ratio in females and males, respectively) but the CC growth was even more impaired (31% and 19% smaller in females and males, respectively) 4 months after IR compared with non-irradiated mice. In conclusion, this is the first study to our knowledge demonstrating that IR to the young rodent brain affects white matter development more in females than in males.

Lipopolysaccharide-Induced Inflammation Aggravates Irradiation-Induced Injury to the Young Mouse Brain

Radiotherapy is an effective treatment strategy in the treatment of brain tumors, but it is also a major cause of long-term complications, especially in survivors of pediatric brain tumors. Cognitive decline caused by cranial radiotherapy is thought, at least partly, to depend on injury to stem and progenitor cells in the dentate gyrus of the hippocampus. This study investigated the effects of lipopolysaccharide (LPS)-induced inflammation at the time of irradiation (IR) in the growing mouse brain. A single injection of LPS (0.3 mg/kg) was administered 24 h prior to cranial IR of 14-day-old male mice. LPS pretreatment increased the levels of the chemokine CCL2 and the cytokine IL-1β in the brain by 440 and 560%, respectively, compared to IR alone. IR disrupted hippocampal neurogenesis and the growth of the dentate gyrus, and the mice pretreated with LPS displayed an even more pronounced lack of growth than the vehicle-treated group 2 months after IR. The density of microglia was not affected, but LPS-pretreated mice displayed 48% fewer bromodeoxyuridine-positive cells and 43% fewer doublecortin-positive cells in the granule cell layer 2 months after IR compared with the vehicle-treated group. In conclusion, an ongoing inflammation in the brain at the time of IR further enhanced the IR-induced loss of neurogenesis, and may aggravate future cognitive deficits in patients treated with cranial radiotherapy.