Radoslaw Rola

Radiation-Induced Cognitive Impairments are Associated with Changes in Indicators of Hippocampal Neurogenesis

Abstract Raber, J., Rola, R., LeFevour, A., Morhardt, D., Curley, J., Mizumatsu, S., VandenBerg, S. R. and Fike, J. R. Radiation-Induced Cognitive Impairments are Associated with Changes in Indicators of Hippocampal Neurogenesis. Radiat. Res. 162, 39–47 (2004). During treatment of brain tumors, some head and neck tumors, and other diseases, like arteriovenous malformations, the normal brain is exposed to ionizing radiation. While high radiation doses can cause severe tissue destruction, lower doses can induce cognitive impairments without signs of overt tissue damage. The underlying pathogenesis of these impairments is not well understood but may involve the neural precursor cells in the dentate gyrus of the hippocampus. To assess the effects of radiation on cognitive function, 2-month-old mice received either sham treatment (controls) or localized X irradiation (10 Gy) to the hippocampus/cortex and were tested behaviorally 3 months later. Compared to controls, X-irradiated mice showed hippocampal-dependent spatial learning and memory impairments in the Barnes maze but not the Morris water maze. No nonspatial learning and memory impairments were detected. The cognitive impairments were associated with reductions in proliferating Ki-67-positive cells and Doublecortin-positive immature neurons in the subgranular zone (SGZ) of the dentate gyrus. This study shows significant cognitive impairments after a modest dose of radiation and demonstrates that the Barnes maze is particularly sensitive for the detection of radiation-induced cognitive deficits in young adult mice. The significant loss of proliferating SGZ cells and their progeny suggests a contributory role of reduced neurogenesis in the pathogenesis of radiation-induced cognitive impairments.

Radiation Response of Neural Precursor Cells: Linking Cellular Sensitivity to Cell Cycle Checkpoints, Apoptosis and Oxidative Stress

Abstract Limoli, C. L., Giedzinski, E., Rola, R., Otsuka, S., Palmer, T. D. and Fike, J. R. Radiation Response of Neural Precursor Cells: Linking Cellular Sensitivity to Cell Cycle Checkpoints, Apoptosis and Oxidative Stress. Radiat. Res. 161, 17–27 (2004). Therapeutic irradiation of the brain can cause a progressive cognitive dysfunction that may involve defects in neurogenesis. In an effort to understand the mechanisms underlying radiation-induced stem cell dysfunction, neural precursor cells isolated from the adult rat hippocampus were analyzed for acute (0–24 h) and chronic (3–33 days) changes in apoptosis and reactive oxygen species (ROS) after exposure to X rays. Irradiated neural precursor cells exhibited an acute dose-dependent apoptosis accompanied by an increase in ROS that persisted over a 3–4-week period. The radiation effects included the activation of cell cycle checkpoints that were associated with increased Trp53 phosphorylation and Trp53 and p21 (Cdkn1a) protein levels. In vivo, neural precursor cells within the hippocampal dentate subgranular zone exhibited significant sensitivity to radiation. Proliferating precursor cells and their progeny (i.e. immature neurons) exhibited dose-dependent reductions in cell number. These reductions were less severe in Trp53-null mice, possibly due to the disruption of apoptosis. These data suggest that the apoptotic and ROS responses may be tied to Trp53-dependent regulation of cell cycle control and stress-activated pathways. The temporal coincidence between in vitro and in vivo measurements of apoptosis suggests that oxidative stress may provide a mechanistic explanation for radiation-induced inhibition of neurogenesis in the development of cognitive impairment.

Alterations in hippocampal neurogenesis following traumatic brain injury in mice

Clinical and experimental data show that traumatic brain injury (TBI)-induced cognitive changes are often manifest as deficits in hippocampal-dependent functions of spatial information processing. The underlying mechanisms for these effects have remained elusive, although recent studies have suggested that the changes in neuronal precursor cells in the dentate subgranular zone (SGZ) of the hippocampus might be involved. Here, we assessed the effects of unilateral controlled cortical impact on neurogenic cell populations in the SGZ in 2-month-old male C57BL6 mice by quantifying numbers of dying cells (TUNEL), proliferating cells (Ki-67) and immature neurons (Doublecortin, Dcx) up to 14 days after TBI. Dying cells were seen 6 h after injury, peaked at 24 h and returned to control levels at 14 days. Proliferating cells were decreased on the ipsilateral and contralateral sides at all the time points studied except 48 h after injury when a transient increase was seen. Simultaneously, immature neurons were reduced up to 84% relative to controls on the ipsilateral side. In the first week post-TBI, reduced numbers of Dcx-positive cells were also seen in the contralateral side; a return to control levels occurred at 14 days. To determine if these changes translated into longer-term effects, BrdU was administered 1 week post-injury and 3 weeks later the phenotypes of the newly born cells were assessed. TBI induced decreases in the numbers of BrdU-positive cells and new neurons (BrdU/NeuN) on the ipsilateral side without apparent changes on the contralateral side, whereas astrocytes (BrdU/GFAP) were increased on the ipsilateral side and activated microglia (BrdU/CD68) were increased on both ipsi- and contralateral sides. No differences were noted in oligodendrocytes (BrdU/NG2). Taken together, these data demonstrate that TBI alters both neurogenesis and gliogenesis. Such alterations may play a contributory role in TBI-induced cognitive impairment.

High-LET radiation induces inflammation and persistent changes in markers of hippocampal neurogenesis.

Abstract Rola, R., Sarkissian, V., Obenaus, A., Nelson, G. A., Otsuka, S., Limoli, C. L. and Fike, J. R. High-LET Radiation Induces Inflammation and Persistent Changes in Markers of Hippocampal Neurogenesis. Radiat. Res. 164, 556–560 (2005). Exposure to heavy-ion radiation is considered a potential health risk in long-term space travel. It may result in the loss of critical cellular components in complex systems like the central nervous system (CNS), which could lead to performance decrements that ultimately could compromise mission goals and long-term quality of life. Specific hippocampal-dependent cognitive impairment occurs after whole-body 56Fe-particle irradiation, and while the pathogenesis of this effect is not yet clear, it may involve damage to neural precursor cells in the hippocampal dentate gyrus. We irradiated mice with 1–3 Gy of 12C or 56Fe ions and 9 months later quantified proliferating cells and immature neurons in the dentate subgranular zone (SGZ). Our results showed that reductions in these cells were dependent on the dose and LET. When compared with data for mice that were studied 3 months after 56Fe-particle irradiation, our current data suggest that these changes are not only persistent but may worsen with time. Loss of precursor cells was also associated with altered neurogenesis and a robust inflammatory response. These results indicate that high-LET radiation has a significant and long-lasting effect on the neurogenic population in the hippocampus that involves cell loss and changes in the microenvironment.

Traumatic injury to the immature brain results in progressive neuronal loss, hyperactivity and delayed cognitive impairments.

The immature brain may be particularly vulnerable to injury during critical periods of development. To address the biologic basis for this vulnerability, mice were subjected to traumatic brain injury at postnatal day 21, a time point that approximates that of the toddler-aged child. After motor and cognitive testing at either 2 weeks (juveniles) or 3 months (adults) after injury, animals were euthanized and the brains prepared for quantitative histologic assessment. Brain-injured mice exhibited hyperactivity and age-dependent anxiolysis. Cortical lesion volume and subcortical neuronal loss were greater in brain-injured adults than in juveniles. Importantly, cognitive decline was delayed in onset and coincided with loss of neurons in the hippocampus. Our findings demonstrate that trauma to the developing brain results in a prolonged period of pathogenesis in both cortical and subcortical structures. Behavioral changes are a likely consequence of regional-specific neuronal degeneration.

Hippocampal Neurogenesis and Neuroinflammation after Cranial Irradiation with 56Fe Particles

Abstract Rola, R., Fishman, K., Baure, J., Rosi, S., Lamborn, K. R., Obenaus, A., Nelson, G. A. and Fike, J. R. Hippocampal Neurogenesis and Neuroinflammation after Cranial Irradiation with 56Fe Particles. Radiat. Res. 169, 626–632 (2008). Exposure to heavy-ion radiation is considered a potential health risk in long-term space travel. In the central nervous system (CNS), loss of critical cellular components may lead to performance decrements that could ultimately compromise mission goals and long-term quality of life. Hippocampal-dependent cognitive impairments occur after exposure to ionizing radiation, and while the pathogenesis of this effect is not yet clear, it may involve the production of newly born neurons (neurogenesis) in the hippocampal dentate gyrus. We irradiated mice with 0.5–4 Gy of 56Fe ions and 2 months later quantified neurogenesis and numbers of activated microglia as a measure of neuroinflammation in the dentate gyrus. Results showed that there were few changes after 0.5 Gy, but that there was a dose-related decrease in hippocampal neurogenesis and a dose-related increase in numbers of newly born activated microglia from 0.5–4.0 Gy. While those findings were similar to what was reported after X irradiation, there were also some differences, particularly in the response of newly born glia. Overall, this study showed that hippocampal neurogenesis was sensitive to relatively low doses of 56Fe particles, and that those effects were associated with neuroinflammation. Whether these changes will result in functional impairments or if/how they can be managed are topics for further investigation.

Indicators of Hippocampal Neurogenesis are Altered by 56Fe-Particle Irradiation in a Dose-Dependent Manner

Abstract Rola, R., Otsuka, S., Obenaus, A., Nelson, G. A., Limoli, C. L., VandenBerg, S. R. and Fike, J. R. Indicators of Hippocampal Neurogenesis are Altered by 56Fe-Particle Irradiation in a Dose-Dependent Manner. Radiat. Res. 162, 442–446 (2004). The health risks to astronauts exposed to high-LET radiation include possible cognitive deficits. The pathogenesis of radiation-induced cognitive injury is unknown but may involve loss of neural precursor cells from the subgranular zone (SGZ) of the hippocampal dentate gyrus. To address this hypothesis, adult female C57BL/6 mice received whole-body irradiation with a 1 GeV/nucleon iron-particle beam in a single fraction of 0, 1, 2 and 3 Gy. Two months later mice were given BrdU injections to label proliferating cells. Subsequently, hippocampal tissue was assessed using immunohistochemistry for detection of proliferating cells and immature neurons. Routine histopathological methods were used to qualitatively assess tissue/cell morphology in the hippocampal formation and adjacent areas. When compared to controls, irradiated mice showed progressively fewer BrdU-positive cells as a function of dose. This observation was confirmed by Ki-67 immunostaining in the SGZ showing reductions in a dose-dependent fashion. The progeny of the proliferating SGZ cells, i.e. immature neurons, were visualized by doublecortin staining and were significantly reduced by irradiation, with the decreases ranging from 34% after 1 Gy to 71% after 3 Gy. Histopathology showed that in addition to cell changes in the SGZ, 56Fe particles induced a chronic and diffuse astrocytosis and changes in pyramidal neurons in and around the hippocampal formation. The present data provide the first evidence that high-LET radiation has deleterious effects on cells associated with hippocampal neurogenesis.

Efficient Production of Reactive Oxygen Species in Neural Precursor Cells after Exposure to 250 MeV Protons

Abstract Giedzinski, E., Rola, R., Fike, J. R. and Limoli, C. L. Efficient Production of Reactive Oxygen Species in Neural Precursor Cells after Exposure to 250 MeV Protons. Radiat. Res. 164, 540–544 (2005). The space radiation environment is composed of highly energetic ions, dominated by protons, that pose a range of potential health risks to astronauts. Traversals of these particles through certain tissues may compromise the viability and/or function of sensitive cells, including neural precursors found within the dentate subgranular zone of the hippocampus. Irradiation has been shown to deplete these cells in vivo, and reductions of these critical cells are believed to impair neurogenesis and cognition. To more fully understand the mechanisms underlying the behavior of these precursor cells after irradiation, we have developed an in vitro neural precursor cell system and used it to assess acute (0–48 h) changes in ROS and mitochondrial end points after exposure to Bragg-peak protons of 250 MeV. Relative ROS levels were increased at nearly all doses (1–10 Gy) and postirradiation times (6–24 h) compared to unirradiated controls. The increase in ROS after proton irradiation was more rapid than that observed with X rays and showed a well-defined dose response at 6 and 24 h, increasing approximately 10% and 3% per gray, respectively. However, by 48 h postirradiation, ROS levels fell below controls and coincided with minor reductions in mitochondrial content. Use of the antioxidant α-lipoic acid (before or after irradiation) was shown to eliminate the radiation-induced rise in ROS levels. Our results corroborate earlier studies using X rays and provide further evidence that elevated ROS are integral to the radioresponse of neural precursor cells.

Altered growth and radiosensitivity in neural precursor cells subjected to oxidative stress

Purpose: To determine whether changes in oxidative stress could enhance the sensitivity of neural precursor cells to ionizing radiation. Materials and methods: Two strategies were used whereby oxidative stress was modulated endogenously, through manipulation cell culture density, or exogenously, through direct addition of hydrogen peroxide. Results: Cells subjected to increased endogenous oxidative stress through low-density growth routinely exhibited an inhibition of growth following irradiation. However, cells subjected to chronic exogenous oxidative treatments showed increased sensitivity to proton and γ-irradiation compared to untreated controls. Reduced survival of irradiated cultures subjected to oxidizing conditions was corroborated using enzymatic viability assays, and was observed over a range of doses (1 – 5 Gy) and post-irradiation re-seeding densities (20 – 200 K/plate). Conclusions: Collectively our results provide further support for the importance of redox state in the regulation of neural precursor cell function, and suggest that oxidative stress can inhibit the proliferative potential of cells through different mechanisms. This is likely to compromise survival and under conditions where excess exogenous oxidants might predominate, sensitivity to irradiation may be enhanced.

Increased tissue perfusion promotes capillary dysplasia in the ALK1-deficient mouse brain following VEGF stimulation

Loss-of-function activin receptor-like kinase 1 gene mutation (ALK1+/-) is associated with brain arteriovenous malformations (AVM) in hereditary hemorrhagic telangiectasia type 2. Other determinants of the lesional phenotype are unknown. In the present study, we investigated the influence of high vascular flow rates on ALK1+/- mice by manipulating cerebral blood flow (CBF) using vasodilators. Adult male ALK1+/- mice underwent adeno-associated viral-mediated vascular endothelial growth factor (AAVVEGF) or lacZ (AAVlacZ as a control) gene transfer into the brain. Two weeks after vector injection, hydralazine or nicardipine was infused intraventricularly for another 14 days. CBF was measured to evaluate relative tissue perfusion. We analyzed the number and morphology of capillaries. Results demonstrated that hydralazine or nicardipine infusion increased focal brain perfusion in all mice. It was noted that focal CBF increased most in AAVVEGF-injected ALK1+/- mice following hydralazine or nicardipine infusion (145+/-23% or 150+/-11%; P<0.05). There were more detectable dilated and dysplastic capillaries (2.4+/-0.3 or 2.0+/-0.4 dysplasia index; P<0.01) in the brains of ALK1+/- mice treated with AAVVEGF and hydralazine or nicardipine compared with the mice treated with them individually. We concluded that increased focal tissue perfusion and angiogenic factor VEGF stimulation could have a synergistic effect to promote capillary dysplasia in a genetic deficit animal model, which may have relevance to further studies of AVMs.