NOTE: End date extended via NCE to 11/28/2009 per PI (3/2009)

End date changed to 2/28/2009 per PI (5/08)

End date changed to 1/23/2009 per JSC info update (7/07)

Foremost in the Critical Path Roadmap system for radiation effects on the central nervous system is Risk #29 – Acute and Late CNS Risks. All of the research and technology questions associated with this risk have priority level one for Mars missions and priority level 3 levels for ISS and 30d lunar missions. The questions addressed by the NSCOR project are 29a (functional changes), 29b (late degeneration), 29c (Alzheimer predisposition), 29e (late degeneration), 29g (cell and tissue mechanisms of damage), 29h (different cell populations including stem cells) and 29i (biomarkers). Also, a NASA workshop on CNS radiobiology identified supporting questions to assist in designing experiments to elucidate CNS effects in response to radiation exposure. These questions include: 1) What is the latency to CNS injury, 2) What cell types are involved in CNS injury (neurons, stem cells, glia)? and 3) What vascular changes occur in response to exposure. We have used this framework to focus our investigation on the effects of charged particles on the CNS. Specifically we will quantify cellular and functional changes in the hippocampus following high energy protons (main SPE constituent) and Fe-56 particles . We will use male rodents with sufficient statistical samples to address inter-individual variation and will address workshop questions 1-3 by examining the responses of different constituent cell types and the latency of damage. NSCOR subproject A quantifies different cell populations (29b, 29e, 29g, 29h); subproject B addresses functional changes and non-invasive techniques for identifying biomarkers (29a, 29i); and subproject C targets late degenerative changes with an Alzheimer model and the role of inflammation (29b, 29c, 29e, 29g and 29i). Our strategy is therefore aligned with the programmatic goals set forth by NASA and its advisory groups.

Aim A1. Significant time and dose dependent progressive loss of endothelial cells are seen along with loss and remodeling of microvessels; hippocampal subfields are differentially affected. This predicts that vascular insufficiency is occurring that may be manifest as poor perfusion and hypoxia with reduced performance of parenchymal cells. Observations for iron ions are recapitulated for animals exposed to protons. However, maximum cell loss is observed after 1 Gray rather than 0.5 Gray. A collaborative effort with University of Arizona Professor Timothy Secomb is allowing us to predict local tissue perfusion using a mathematical model developed by Dr. Secomb.

Aim A2. Significant time and dose dependent loss of neural precursor cells is observed in a lineage-specific pattern. Loss of neural precursor cells is associated with cognitive impairment and spatial memory problems.

Aim A3. Total microglial cell numbers are relatively unaffected by irradiation but sharp increases in newly-born microglia occur post-irradiation. Different phenotypic changes are seen in cells which are perivascular versus those that are not. This suggests that microglia are responding to persistent radiation-induced inflammatory changes or alterations to the microenvironment.

Aim A4. Assessment of DNA damage in the hippocampi of transgenic mice shows that mutations continue to accumulate with time after irradiation indicating the continued presence of latent DNA damage. Their structural spectrum is LET and time dependent and differs from whole brain indicating regional specialization in adaptation to genotoxicity Gene expression profiling demonstrates activation of neurotrophic and adhesion factors associated with synaptic plasticity as well as up-regulation of chemokine receptors associated with inflammatory processes.

Aims A1-4. Methods Summary: Perfusion Fixation/Euthanasia, Immunohistochemistry, Confocal Image acquisition, Gene expression studies, Mutation Analysis.

Aim B1. Extracellular recordings from the CA1 region evaluated pyramidal cells after stimulation of CA3 axons. Input-output tests demonstrated time and dose dependent modifications to both dendritic and somatic region properties that are associated with enhanced excitability and decreased synaptic efficacy. Long-term potentiation protocols demonstrated a reduction of synaptic plasticity following irradiation that is consistent with a potential reduction of memory forming ability. In vitro patch clamp recordings showed that intrinsic membrane properties of neurons are resistant to radiation. Pharmacological manipulations then demonstrated changes in the inhibitory synapses on CA1 pyramidal cells suggesting that their hyperexcitability may be due in part to a reduction in GABAergic inhibition. Dysregulation of inhibition could be associated with seizure risk.

Aim B2. Diffusion Tensor Imaging visualized and quantified altered structural brain changes based on altered water diffusion properties (direction, magnitude and anisotropy). Alterations in the corpus callosum, exterior capsule and hippocampus were observed and histological comparisons are underway to determine whether early aspects of demyelination or gliosis are being detected.

Aims B1-3. Methods Summary: In Vitro Brain Slice Preparation, Extracellular Recordings and Long Term Potentiation (LTP), Patch Clamp Recordings, Histological Assessment, Diffusion Tensor Imaging.

Aim C1. APP23 transgenic mice recreate most of the pathological features of Alzheimer's Disease, and most importantly, the disease progression is continuous over several months. These mice also feature prominent alterations of the vasculature, presumably one of the most vulnerable tissues in the brain after radiation. Electrophysiological measurements are showing that disease-related decreases in neuronal output and synaptic efficacy occur earlier in irradiated animals, which confirms our original hypothesis. This is consistent with the idea that that radiation may accelerate late neurogenerative changes . Using 3D vascular polymer cast technology combined with synchrotron based tomographic microscopy, vascular topology changes after irradiation have been detected. For the hippocampus, these changes are consistent with a change in vessel segment length and decreased spacing between vessels. As in Aim A1 this may be indicative of reduced perfusion and function of parenchymal cells. We expect to observe neovascularization and infarcts in older APP mice and will compare the time course of this progression in control and irradiated animals to further test whether radiation causes a decreased latency in disease progression. Amyloid Beta plaque load is being assessed using ThioS staining and preliminary results indicate an acceleration of plaque formation following irradiation.

Aim C2. In order to assess the ability of the brain to respond to external environmental shocks and restore orderly normal function (homeostasis) we apply a controlled septic shock to cause an inflammatory reaction mediated by the periphery--nerves and immune system. The CNS response to peripheral lipopolysaccharide (LPS) exposure has long been established and typically includes changes in electrophysiology and cytokine expression. We found that in irradiated animals, the patterns of electrophysiological changes associated with reactions to LPS are complex and unlike those of either LPS or irradiation alone. We had expected the effects to be additive or synergistic. This indicates that radiation causes plastic changes to the CNS that persist for many months in the mouse and alter its program of response to septic shock. This is consistent with the idea that irradiation may potentiate the risks from late secondary insults which might include trauma or infection.

Aims C1-C2. Methods Summary: In vitro electrophysiology as in Aim B1, Extracellular Protocols and Analysis of Neuronal Activity, Corrosion casting, Synchrotron X-ray microCT (XTM), Confocal microscopy, Image analysis, LPS Administration.

Complementary experiments assessing mechanisms and establishing baseline for new NSCOR project.

Administration of fluorescent hypoxia marker piminidazolento irradiated animals demonstrated dose and time dependent hypoxic status in hippocampus in regional patterns matching microvessel sensitivity. This supports the idea that at higher doses a hypoxic episode evokes neoangiogenesis.

A microelectrode array system (Panasonic MED64) has been installed and used successfully to simultaneously record from multiple sites in the hippocampus circuitry allowing characterization of network-level activity. A hypoxia reperfusion protocol with irradiated hippocampal slices has shown an interaction between tissue responses to oxidative stress and radiation.

Electrophysiological experiments with geriatric irradiated rats shows age-related sensitivity to charged particles for electrophysiology parameters including long term potentiation. Rat electrophysiology responses parallel those in mice.

Eyes of geriatric irradiated rats were analyzed for late degeneration using diffusion tensor imaging and exhibited retinal and optic nerve changes.

Proceedings, World Congress 2009, Medical Physics and Biomedical Engineering, Munich, Germany, Sept 7-12, 2009. , Sep-2009

Online Abstract. NASA Human Research Program Investigators’ Workshop, League City, TX, February 2-4, 2009. http://www.dsls.usra.edu/meetings/hrp2009/pdf/CNSDegenerativeRisks/1115Nelson.pdf , Feb-2009

Heavy Ions in Therapy and Space Symposium 2009, Cologne, Germany, July 6-10, 2009. , Jul-2009

Proceedings. Microscopy Conference 2009, Graz, Austria, Aug 30-Sep 4, 2009. , Jun-2009

Abstract Book. 55th Annual Meeting of the Radiation Research Society, Savannah, GA, October 3-7, 2009. , Oct-2009

Proceedings. Heavy Ions in Therapy and Space Symposium 2009, Cologne, Germany, July 6-10, 2009. , Jul-2009

Proceedings. 12th Annual Force Health Protection Conference, Albuquerque, NM, August 14-21, 2009. , Aug-2009

Proceedings. 17th Scientific Meeting, International Society for Magnetic Resonance in Medicine, Honolulu, Hawaii, April 18-24, 2009. , Aug-2009

Proceedings. 17th Scientific Meeting, International Society for Magnetic Resonance in Medicine, Honolulu, Hawaii, April 18-24, 2009. , Aug-2009

Proceedings. Heavy Ions in Therapy and Space Symposium 2009, Cologne, Germany, July 6-10, 2009. , Jul-2009

Proceedings of the 11th International Congress of the International Union for Physical and Engineering Sciences in Medicine. World Congress 2009. Medical Physics and Biomedical Engineering. ISSN 1680-0737 , May-2009

NOTE: End date extended via NCE to 11/28/2009 per PI (3/2009)

End date changed to 2/28/2009 per PI (5/08)

End date changed to 1/23/2009 per JSC info update (7/07)

Foremost in the Critical Path Roadmap system for radiation effects on the central nervous system is Risk #29 – Acute and Late CNS Risks. All of the research and technology questions associated with this risk have priority level one for Mars missions and priority level 3 levels for ISS and 30d lunar missions. The questions addressed by the NSCOR project are 29a (functional changes), 29b (late degeneration), 29c (Alzheimer predisposition), 29e (late degeneration), 29g (cell and tissue mechanisms of damage), 29h (different cell populations including stem cells) and 29i (biomarkers). Also, a NASA workshop on CNS radiobiology identified supporting questions to assist in designing experiments to elucidate CNS effects in response to radiation exposure. These questions include: 1) What is the latency to CNS injury, 2) What cell types are involved in CNS injury (neurons, stem cells, glia)? and 3) What vascular changes occur in response to exposure. We have used this framework to focus our investigation on the effects of charged particles on the CNS. Specifically we will quantify cellular and functional changes in the hippocampus following high energy protons (main SPE constituent) and Fe-56 particles . We will use male rodents with sufficient statistical samples to address inter-individual variation and will address workshop questions 1-3 by examining the responses of different constituent cell types and the latency of damage. NSCOR subproject A quantifies different cell populations (29b, 29e, 29g, 29h); subproject B addresses functional changes and non-invasive techniques for identifying biomarkers (29a, 29i); and subproject C targets late degenerative changes with an Alzheimer model and the role of inflammation (29b, 29c, 29e, 29g and 29i). Our strategy is therefore aligned with the programmatic goals set forth by NASA and its advisory groups.

Aim A1. Significant time and dose dependent progressive loss of endothelial cells are seen along with loss and remodeling of microvessels; hippocampal subfields are differentially affected. This predicts that vascular insufficiency is occurring that may be manifest as poor perfusion and hypoxia with reduced performance of parenchymal cells. Observations reported previously for iron ions are being recapitulated for animals exposed to protons. However, maximum cell loss is observed after 1 Gray rather than 0.5 Gray. A collaborative effort with University of Arizona Professor Timothy Secomb is allowing us to predict local tissue perfusion using a mathematical model developed by Dr. Secomb.

Aim A2. Significant time and dose dependent loss of neural precursor cells is observed in a lineage-specific pattern. Loss of neural precursor cells is associated with cognitive impairment and spatial memory problems.

Aim A3. Total microglial cell numbers are relatively unaffected by irradiation but sharp increases in newly-born microglia occur post-irradiation. Different phenotypic changes are seen in cells which are perivascular versus those that are not. This suggests that microglia are responding to persistent radiation-induced inflammatory changes or alterations to the microenvironment.

Aim A4. Assessment of DNA damage in the hippocampi of transgenic mice shows that mutations continue to accumulate with time after irradiation indicating the continued presence of latent DNA damage. Their structural spectrum is LET and time dependent and differs from whole brain indicating regional specialization in adaptation to genotoxicity Gene expression profiling demonstrates activation of neurotrophic and adhesion factors associated with synaptic plasticity as well as up-regulation of chemokine receptors associated with inflammatory processes.

Aims A1-4. Methods Summary: Perfusion Fixation/Euthanasia, Immunohistochemistry, Confocal Image acquisition, Gene expression studies, Mutation Analysis.

Aim B1. Extracellular recordings from the CA1 region evaluated pyramidal cells after stimulation of CA3 axons. Input-output tests demonstrated time and dose dependent modifications to both dendritic and somatic region properties that are associated with enhanced excitability and decreased synaptic efficacy. Long-term potentiation protocols demonstrated a reduction of synaptic plasticity following irradiation that is consistent with a potential reduction of memory forming ability.

In vitro sharp electrode and patch clamp recordings showed that intrinsic membrane properties of neurons are resistant to radiation. Pharmacological manipulations then demonstrated changes in the inhibitory synapses on CA1 pyramidal cells suggesting that their hyperexcitability may be due in part to a reduction in GABAergic inhibition. Dysregulation of inhibition could be associated with seizure risk.

Aim B2. Diffusion Tensor Imaging visualized and quantified altered structural brain changes based on altered water diffusion properties (direction, magnitude and anisotropy). Alterations in the corpus callosum, exterior capsule and hippocampus were observed and histological comparisons are underway to determine whether early aspects of demyelination or gliosis are being detected.

Aims B1-3. Methods Summary: In Vitro Brain Slice Preparation, Extracellular Recordings and Long Term Potentiation (LTP), Patch Clamp Recordings, Histological Assessment, Diffusion Tensor Imaging.

Aim C1. APP23 transgenic mice recreate most of the pathological features of Alzheimer’s Disease, and most importantly, the disease progression is continuous over several months. These mice also feature prominent alterations of the vasculature, presumably one of the most vulnerable tissues in the brain after radiation. Electrophysiological measurements are showing that disease-related decreases in neuronal output and synaptic efficacy occur earlier in irradiated animals –which confirms our original hypothesis. This is consistent with the idea that that radiation may accelerate late neurogenerative changes.

Using 3D vascular polymer cast technology combined with synchrotron based tomographic microscopy, vascular topology changes after irradiation have been detected. For the hippocampus, these changes are consistent with a change in vessel segment length and decreased spacing between vessels. As in Aim A1 this may be indicative of reduced perfusion and function of parenchymal cells. We expect to observe neovascularization and infarcts in older APP mice and will compare the time course of this progression in control and irradiated animals to further test whether radiation causes a decreased latency in disease progression. Amyloid Beta plaque load is being assessed using ThioS staining and preliminary results indicate an acceleration of plaque formation following irradiation.

Aim C2. In order to assess the ability of the brain to respond to external environmental shocks and restore orderly normal function (homeostasis) we apply a controlled septic shock to cause an inflammatory reaction mediated by the periphery – nerves and immune system. The CNS response to peripheral lipopolysaccharide (LPS) exposure has long been established and typically includes changes in electrophysiology and cytokine expression. We found that in irradiated animals, the patterns of electrophysiological changes associated with reactions to LPS are complex and unlike those of either LPS or irradiation alone. We had expected the effects to be additive or synergistic. This indicates that radiation causes plastic changes to the CNS that persist for many months in the mouse and alter its program of response to septic shock. This is consistent with the idea that irradiation may potentiate the risks from late secondary insults which might include trauma or infection.

Aims C1-C2. Methods Summary: In vitro electrophysiology as in Aim B1, Extracellular Protocols and Analysis of Neuronal Activity, Corrosion casting, Synchrotron X-ray microCT (XTM), Confocal microscopy, Image analysis, LPS Administration.

11th International Congress of the International Union for Physical and Engineering Sciences in Medicine. World Congress 2009. Medical Physics and Biomedical Engineering, Munich, Sept 7-12, 2009. , Sep-2009

Space Life Sciences Seminar, Texas A&M University, College Station, TX, March 11, 2009. , Mar-2009

Abstract Book. NASA Human Research Program Investigators’ Workshop, League City, TX, February 2-4, 2009. , Feb-2009

Dean’s Research Seminar, San Francisco General Hospital, (UCSF), December, 2008. , Dec-2008

Abstract Book. Society for Neuroscience Meeting, Washington, D.C., November 18, 2008. , Nov-2008

Abstract Book. Society for Neuroscience Meeting, Washington, D.C., November 18, 2008. , Nov-2008

Seminar. Neural Injury and Repair Series, Neonatal Brain Disorders Center & Brain and Spinal Injury Center, UCSF, November, 2008. , Nov-2008

Seminar. Nevada Cancer Institute, Las Vegas, NV, October, 2008. , Oct-2008

Abstract Book. 54th Annual Radiation Research Society Meeting, Boston, Sept. 21-24, 2008. , Sep-2008

Abstract Book. 9th International Conference on X-Ray Microscopy, Zürich, Switzerland, July 21 - 25, 2008. , Jul-2008

Proceedings. 37th COSPAR Scientific Assembly, Montreal, July 13-20, 2008. , Jul-2008

Abstract Book. 19th Annual NASA Space Radiation Investigators’ Workshop, Philadelphia, PA, June 30 – July 2, 2008. , Jul-2008

Abstract Book , Jul-2008

Abstract Book , Jul-2008

Proceedings , Feb-2008

Proceedings. NASA Human Research Program Investigators’ Workshop, League City, TX, February 4-6, 2008. , Feb-2008

Proceedings. NASA Human Research Program Investigators’ Workshop, League City, TX, February 4-6, 2008. , Feb-2008

Proceedings. NASA Human Research Program Investigators’ Workshop, League City, TX, February 4-6, 2008. , Feb-2008

Proceedings of the 11th International Congress of the International Union for Physical and Engineering Sciences in Medicine. World Congress 2009. Medical Physics and Biomedical Engineering, Munich, Sept 7-12, 2009. Springer Verlag. ISSN 1680-0737, in press July 2009. , Jul-2009

End date changed to 2/28/2009 per PI (5/08)

End date changed to 1/23/2009 per JSC info update (7/07)

Foremost in the Critical Path Roadmap system for radiation effects on the central nervous system is Risk #29 – Acute and Late CNS Risks. All of the research and technology questions associated with this risk have priority level one for Mars missions and priority level 3 levels for ISS and 30d lunar missions. The questions addressed by the NSCOR project are 29a (functional changes), 29b (late degeneration), 29c (Alzheimer predisposition), 29e (late degeneration), 29g (cell and tissue mechanisms of damage), 29h (different cell populations including stem cells) and 29i (biomarkers). Also, a NASA workshop on CNS radiobiology identified supporting questions to assist in designing experiments to elucidate CNS effects in response to radiation exposure. These questions include: 1) What is the latency to CNS injury, 2) What cell types are involved in CNS injury (neurons, stem cells, glia)? and 3) What vascular changes occur in response to exposure. We have used this framework to focus our investigation on the effects of charged particles on the CNS. Specifically we will quantify cellular and functional changes in the hippocampus following high energy protons (main SPE constituent) and Fe-56 particles . We will use male rodents with sufficient statistical samples to address inter-individual variation and will address workshop questions 1-3 by examining the responses of different constituent cell types and the latency of damage. NSCOR subproject A quantifies different cell populations (29b, 29e, 29g, 29h); subproject B addresses functional changes and non-invasive techniques for identifying biomarkers (29a, 29i); and subproject C targets late degenerative changes with an Alzheimer model and the role of inflammation (29b, 29c, 29e, 29g and 29i). Our strategy is therefore aligned with the programmatic goals set forth by NASA and its advisory groups.

Space Radiation

The unique feature of the space radiation environment is the presence of high-energy charged particles dominated by protons from the sun and a complex spectrum of galactic cosmic rays consisting of ionized nuclei of stable elements. Of these, protons are estimated to contribute 19% of the dose equivalent in space while 56Fe ions constitute 13% and carbon and oxygen each contribute about 6 %. Estimated radiation exposures for long term missions are estimated to be of the order of 1 – 2 Sieverts.

CNS Radiobiology

The brain is the site of executive control over homeostatic mechanisms via electrical and endocrine signaling networks, and therefore of critical importance to all other organ and tissue systems under its coordination. While high radiation doses to the brain result in significant tissue destruction (e.g. demyelination and necrosis), subtle changes can occur that may have significant physiologic impact. Such changes could involve loss of critical cell populations such as neural precursor cells, neurons, and glia or alterations in the microvasculature. Microenvironment changes might result in dysregulation of synaptic plasticity and electrical signal processing. Little data are available radiation reactions of CNS at low doses and with charged particles which will determine the risks associated with protracted charged particle exposures in space.

Hypotheses

With these CNS and radiation features in mind, we established a main hypothesis and a set of three specific hypotheses to organize our experimental strategy. The main hypothesis is: Charged particle radiation exposure induces progressive deleterious structural and functional changes in the brain, leading to performance decline. This leads to the following specific hypotheses and associated aims:

Hypothesis (A) Charged particle irradiation causes a progressive loss of cells and a remodeling of CNS tissue as a function of dose, time and LET. To test hypothesis A we: Specific Aim A1. Quantify the cellular composition of mouse brain vascular endothelium using stereological analysis of preserved tissue. Specific Aim A2. Quantify the composition of mouse neural precursor cells using stereological analysis of preserved tissue. Specific Aim A3. Quantify the activation of microglial cells using stereological analysis immune-labeled tissue. Specific Aim A4. Quantify key molecular biomarkers associated with radiation-induced damage using targeted gene expression profiling and mutation analysis of a structural gene.

Hypothesis (B) Charged particle irradiation alters the functional output or performance of the brain as a function of dose, time and LET. To test hypothesis B we: Specific Aim B1. Quantify electrophysiological properties of mouse brain using tissue slices that represent intact regional ensembles of interacting cells. (extracellular, patch clamp) Specific Aim B2. Map the time evolution of macroscopic structural features in mouse brains using Magnetic Resonance Imaging.

Hypothesis (C) Exposure to radiation decreases neurodegenerative disease latency and alters the response to acute stressors. To test hypothesis C we: Specific Aim C1. Quantify the magnitude and time course of changes in brains of mice manipulated by a mutation that produces an Alzheimer disease-like pathology and limits output ( electrophysiology, Aß plaque load, vessel topology). Specific Aim C2. Quantify the electrophysiological performance of brain slices in mice reacting to the application of systemic immune stress using lipopolysaccharide as a surrogate infectious agent.

Animal Models

The male C57/BL6 mouse is being used for experiments. It was chosen based on its suitability for specific experimental protocols, general reliability in predicting human responses, and wealth of published radiobiological data. Two genetic variants of the C57/BL6 mouse will be used in addition to wild type: an APP (amyloid precursor protein) transgenic strain, APP 23 tg locus and a transgenic variant containing an integrated shuttle vector array with the E. coli ß-galactosidase gene (lacZ).

Irradiation Considerations

To investigate the contributions of the two most important and qualitatively different particles, NSCOR experiments utilize doses of 0.5 to 4 Gy for 56Fe ions and 0-8 Gy for protons. We selected: 250 MeV/amu 1H1+ (LET 0.4 keV/µm) and 600 MeV/amu 56Fe26+ (LET 175 keV/µm). The 250 MeV energy for protons is typical of space radiation and for radiotherapy at Loma Linda University where proton beams are obtained. For iron, 600 MeV/amu was selected to maximize LET, while minimizing fragmentation and facilitating use of a collimator to provide brain-only irradiation. Iron beams are obtained from the NASA Space Radiation Laboratory (NSRL) at the Brookhaven National Laboratory.

Key Observations and Significance

Aim A1

Significant time and dose dependent progressive loss of endothelial cells are seen along with loss and remodeling of microvessels; hippocampal subfields are differentially affected. This predicts that vascular insufficiency is occuring that may be manifest as poor perfusion and hypoxia with reduced performance of parenchymal cells.

Aim A2

Significant time and dose dependent loss of neural precursor cells is observed in a lineage-specific pattern. Loss of neural precursor cells is associated with cognitive impairment and spatial memory problems.

Aim A3

Total microglial cell numbers are relatively unaffected by irradiation but sharp increases in newly-born microglia occur post-irradiation. Different phenotypic changes are seen in cells which are perivascular versus those that are not. This suggests that microglia are responding to persistent radiation-induced inflammatory changes or alterations to the microenvironment.

Aim A4

Assessment of DNA damage in the hippocampi of transgenic mice shows that mutations continue to accumulate with time after irradiation indicating the continued presence of latent DNA damage. Their structural spectrum is LET and time dependent and differs from whole brain indicating regional specialization in adaptation to genotoxicity Gene expression profiling demonstrates activation of neurotrophic and adhesion factors associated with synaptic plasticity as well as up-regulation of chemokine receptors associated with inflammatory processes.

Aim B1

Extracellular recordings from the CA1 region evaluated pyramidal cells after stimulation of CA3 axons. Input-output tests demonstrated time and dose dependent modifications to both dendritic and somatic region properties that are associated with enhanced excitability and decreased synaptic efficacy. Long-term potentiation protocols demonstrated a reduction of synaptic plasticity following irradiation that is consistent with a potential reduction of memory forming ability.

In vitro sharp electrode and patch clamp recordings showed that intrinsic membrane properties of neurons are resistant to radiation. Pharmacological manipulations then demonstrated changes in the inhibitory synapses on CA1 pyramidal cells suggesting that their hyperexcitability may be due in part to a reduction in GABAergic inhibition. Dysregulation of inhibition could, in principle, be associated with risks of seizures.

Aim B2

Diffusion Tensor Imaging visualized altered structural brain changes based on altered water diffusion properties (direction, magnitude and anisotropy). Alterations in the corpus callosum, exterior capsule and hippocampus were observed and histological comparisons are underway to determine whether early aspects of demyelination or gliosis are being detected.

Aim C1

APP23 transgenic mice recreate most of the pathological features of Alzheimer’s Disease, and most importantly, the disease progression is continuous over several months. These mice also feature prominent alterations of the vasculature, presumably one of the most vulnerable tissues in the brain after radiation. Electrophysiological measurements are showing that disease-related decreases in neuronal output and synaptic efficacy occur earlier in irradiated animals –which confirms our original hypothesis. This is consistent with the idea that that radiation may accelerate late neurogenerative changes in animals or people.

Using 3D vascular polymer cast technology combined with 3D-imaging, microvasculature changes following irradiation have been detected and are consistent with loss of vessels and an increased spacing between them. As in Aim A1 this may be indicative of reduced perfusion and function of parenchymal cells. We expect to observe neovascularization and infarcts in older APP mice and will compare the time course of this progression in control and irradiated animals to further test whether radiation causes a decreased latency in disease progression.

Aim C2

In order to assess the ability of the brain to respond to external environmental shocks and restore orderly normal function (homeostasis) we apply a controlled septic shock to cause an inflammatory reaction mediated by the periphery – nerves and immune system. The CNS response to peripheral lipopolysaccharide (LPS) exposure has long been established and typically includes changes in electrophysiology and cytokine expression. We found that in irradiated animals, the patterns of electrophysiological changes associated with reactions to LPS are complex and unlike those of either LPS or irradiation alone. We had expected the effects to be additive or synergistic. This indicates that radiation causes plastic changes to the CNS that persist for many months in the mouse and alter its program of response to septic shock. This is consistent with the idea that irradiation may potentiate the risks from late secondary insults which might include trauma or infection.

European Winter Conference on Brain Research, Villars, Switzerland, March 2007. , Mar-2007

NASA Models of Space Radiation Risks Workshop, Dallas, TX, March 6-7, 2007. , Mar-2007

NASA CNS Risk Workshop, Houston, TX, Oct. 30-31, 2007. , Oct-2007

8th SLS Users Meeting, Paul Scherrer Institut, Villigen, Switzerland, 11.-12.9.2007. , Dec-2007

2007 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, November 2007. , Nov-2007

2007 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2007. Program No. 207.13. , Nov-2007

19th Annual NASA Space Radiation Investigators’ Workshop, Philadelphia, PA, June 30 –July 3, 2008. , Jul-2008

CIMST Symposium 2008, Imaging: Pushing the Limits in Biomedical Research, Zürich, February 5-6, 2008. , Feb-2008

19th Annual NASA Space Radiation Investigators’ Workshop, Philadelphia, PA, June 30 –July 3, 2008. , Jul-2008

9th International Conference on X-Ray Microscopy, Zürich, Switzerland, July 21 - 25, 2008. , Jul-2008

19th Annual NASA Space Radiation Investigators’ Workshop, Philadelphia, PA, June 30 –July 3, 2008. , Jul-2008

NASA Human Research Program Investigators’ Workshop, League City, Texas, February 4-6, 2008. , Feb-2008

End date changed to 1/23/2009 per JSC info update (7/07)

Foremost in the Critical Path Roadmap system for radiation effects on the central nervous system is Risk #29 – Acute and Late CNS Risks. All of the research and technology questions associated with this risk have priority level one for Mars missions and priority level 3 levels for ISS and 30d lunar missions. The questions addressed by the NSCOR project are 29a (functional changes), 29b (late degeneration), 29c (Alzheimer predisposition), 29e (late degeneration), 29g (cell and tissue mechanisms of damage), 29h (different cell populations including stem cells) and 29i (biomarkers). Also, a NASA workshop on CNS radiobiology identified supporting questions to assist in designing experiments to elucidate CNS effects in response to radiation exposure. These questions include: 1) What is the latency to CNS injury, 2) What cell types are involved in CNS injury (neurons, stem cells, glia)? and 3) What vascular changes occur in response to exposure. We have used this framework to focus our investigation on the effects of charged particles on the CNS. Specifically we will quantify cellular and functional changes in the hippocampus following high energy protons (main SPE constituent) and Fe-56 particles . We will use male rodents with sufficient statistical samples to address inter-individual variation and will address workshop questions 1-3 by examining the responses of different constituent cell types and the latency of damage. NSCOR subproject A quantifies different cell populations (29b, 29e, 29g, 29h); subproject B addresses functional changes and non-invasive techniques for identifying biomarkers (29a, 29i); and subproject C targets late degenerative changes with an Alzheimer model and the role of inflammation (29b, 29c, 29e, 29g and 29i). Our strategy is therefore aligned with the programmatic goals set forth by NASA and its advisory groups.

Two significant administrative changes occurred during project year 3. First, an Aim B subtask has been terminated along with the associated sub-contract to develop MRI sequences enabling the mapping of brain metabolic levels indicated by the relative levels of oxy- vs. deoxyhemoglobin. The sequences were established on a clinical system, then successfully rewritten for a Brucker clinical system and transferred to Loma Linda. However, the contractor was unable, in a timely fashion, to configure the software for our Brucker 4.7T and 11.7 T micro-imagers needed for the project. As irradiated animals would no longer be available for imaging before the final software was completed it was decided to terminate the task. The second significant change was the completion of sub-contractual arrangements for the Paul Scherrer Institute in Villigen, Switzerland to provide synchrotron micro-tomography services for 3-D reconstruction of brain vascular corrosion casts. The task officially began in November 2006 under the direction of Dr. Marco Stampanoni.

Technical Progress

Irradiations: Most of the animals scheduled for iron irradiation at the Brookhaven National Laboratory have now been completed and are aging to their appropriate harvest dates. An additional group of transgenic animals are scheduled for irradiation at NSRL on October 17, 2007. All Aim B animals have been irradiated with both iron ions and protons. Proton irradiations of animals for stereological analysis under Aim A were completed in three campaigns: 12/13/06, 1/9/07 and 1/13/07. A final group of animals for mutagenesis experiments (Aim A4) will be irradiated in late summer / early fall 2007 as determined by the breeding schedule of transgenic LacZ mice.

Core Activities: Tissue processing, stereological and immunohistochemical analyses continue under the supervision of Dr. C. Favre. Methods have continuously improved and staff have been trained to use a new Olympus FV1000 Confocal Microscope system that complements an existing Biorad system. This has enhanced the throughput of image acquisition from prepared samples. Several neurogenesis-related samples (Aim A2) have been repeated to eliminate interference on image quality due to tomato lectin labeling for lumens of microvessels. Vascular corrosion casting training was completed and an LLU team is now producing vascular corrosion casts which are being imaged by X-ray micro-tomography at the Paul Scherrer Institute. The central database is operating stably using the Linux operating system in its permanent location.

Aim A Activities.

Microvasculature Stereology. Stereological analysis of iron-irradiated mice is continuing on the set of 230 brains which have been preserved and archived. The systematic sections cut from each brain slice containing compartments of interest were stained to identify the capillaries, and cell nuclei of interest. Microvessel endothelial cell population numbers as well as vessel lengths in CA1 and dentate gyrus (DG) regions of the hippocampus for times up to 12 months following 56Fe have been measured with no significant reduction of endothelial cell density. However a U-shaped dose response curve is emerging for vessel density at 12 months post irradiation possibly reflecting a repopulation by endothelial cells after higher doses.

Neurogenesis. New sets of animals for neurogenesis have been irradiated with iron ions and analyzed by J. Fike et al. at U.C. San Francisco. This set of animals was a repeat of previous exposures but the signal to noise ratio for neuroblast quantitation was improved by elimination of dual labeling with lectin (used to stain microvessel lumens). These counts have been completed and dose dependent reductions in neuron, astrocyte and oligodendrocyte lineages have been observed. In contrast, an increase in newly-born activated microglia was observed.

Biomarkers of Genotoxicity. For assessment of DNA damage and repair, hippocampal tissues were isolated from animals exposed to a range of iron doses at 1, 8 and 16 weeks after irradiation and processed for lacZ transgene mutant frequencies (MF). We observed a trend of increasing MF with increasing radiation dose up to 4 Gy. Analyses of characteristics of mutants obtained from the 8 week samples using restriction fragment length polymorphism (RFLP) shows that the proportions of mutants with size changes and insertions (versus point mutations) changes substantially with dose and suggests that only cells containing small changes in the genome continue to survive after higher doses. Targeted gene expression studies have highlighted time and dose-related changes in the neural cell adhesion molecule (NCAM) and brain derived neurotrophic factor (BDNF) which are involved in synaptogenesis and neurogenesis.

Aim B activities.

Electrophysiology: Extracellular recordings on 400 um thick hippocampal sections have now been obtained for times up to 12 mo. after iron irradiation. Extracellular recordings were performed in the CA1 dendritic region and after input-output curve characterization, long term potentiation (LTP) was induced by tetanic stimulation with recordings for 90 min post LTP induction. Hippocampal LTP was readily induced in 0 Gy animals but this contrasts with head-only irradiated animals at 1 –12 mo. These animals exhibited dose-dependent decrements in post-tetanic potentiation (PTP) and LTP was decreased significantly at 2 and 4 Gy. These results demonstrate that hippocampal plasticity is altered after 56Fe head-only irradiation. Patch clamp analysis of individual nerves has been carried out at UCLA for animals at 3, 6 and 12 months post irradiation but no obvious changes in intrinsic membrane properties were detected. Neuropharmacological tests are now planned to investigate aspects of neurochemical transmission and receptor function.

Magnetic Resonance Imaging. Diffusion tensor imaging (DTI) magnetization sequences and data analysis software have now been transferred to LLU from Washington University and animals are now imaged on-site. Programs for DTI map calculation were applied to water phantom images to optimize signal to noise ratios and to validate custom coil performance. In vivo and ex vivo images have now been acquired for irradiated animals and show changes in fiber tract morphology and function as reported by the relative anisotropy index. Animal cohorts are being aged appropriately for later image acquisition.

BOLD imaging sequences that portray cerebral metabolic rate of oxygen utilization were adapted from a clinical Siemens system to a clinical Brucker MRI system in use at LLU but the software components were not adapted to the systems required for microimaging of mice. The delays in BOLD imaging implementation precluded their use on aging, irradiated animals so the subcontract was not renewed and this task was terminated.

Aim C Activities.

Electrophysiology. In immunologically-challenged mice (lipopolysaccharide teatment) the most significant finding was the inhibitory effect of radiation on excitability of CA1 neurons. This was detected as a decrease of the amplitude of population spikes evoked at maximal stimulation intensity. Paradoxically, in mice previously exposed to iron ions at 0 to 4 Gy, the effects of irradiation abrogated the LPS-induced response suggesting that irradiation alters the ability of hippocampal structures to respond appropriately to the immune stresses. These observations are consistent for times up to 12 months post-irradiation and are maximal at 2 Gy Corrosion Casting Vascular corrosion casting of transgenic mice began in September 2006 following on-site training at LLU. Corrosion casts of APP23 mouse brains are now routinely made and processed with an X-ray contrast agent. Casts have been shipped to the Paul Scherrer Institute whole brain and local 3-D computed tomography maps of the brain vasculature have been made for younger animal cohorts. Regions of interest have been selected for high resolution mapping and data sets for biological analysis will soon be available for analysis at LLU.

18th Annual NASA Space Radiation Investigators’ Workshop, Abstract Book, p 3, July 2007. , Jul-2007

Proceedings, NASA Models of Space Radiation Risks Workshop, Dallas, TX, March 6-7, 2007. , Mar-2007

Proceedings, DAIT, NAID, NIH Medical Countermeasures against Combined Injury Workshop, March 2007. , Mar-2007

Proceedings, Symposium on Neuroprotection and Neurorepair; From Pharmacology to Stem Cells, May 2007. , May-2007

18th Annual NASA Space Radiation Investigators’ Workshop, Abstract Book, p 152, July 2007. , Jul-2007

Proceedings, 13th International Congress of Radiation Research, Abstract Book, p 33, July 2007. , Jul-2007

18th Annual NASA Space Radiation Investigators’ Workshop, Abstract Book, p 90, July 2007. , Jul-2007

18th Annual NASA Space Radiation Investigators’ Workshop, Abstract Book p 91, July 2007. , Jul-2007

Proceedings, NASA Models of Space Radiation Risks Workshop, March 2007. , Mar-2007

Proceedings, 13th International Congress of Radiation Research, Abstract Book, p 22, July 2007. , Jul-2007

18th Annual NASA Space Radiation Investigators’ Workshop, Abstract Book, p 2, July 2007. , Jul-2007

Proceedings, European Winter Conference on Brain Research, February 2007. , Feb-2007

18th Annual NASA Space Radiation Investigators’ Workshop, Absract Book p 96-97, July 2007. , Jul-2007

Program proceedings, Society for Neuroscience, Program # 503.9, October 2006. , Oct-2006

Program proceedings, Society for Neuroscience, Program # 271.15, October 2006. , Oct-2006

18th Annual NASA Space Radiation Investigators’ Workshop, Abstract Book, p 18, July 2007. , Jul-2007

Foremost in the Critical Path Roadmap system for radiation effects on the central nervous system is Risk #29 – Acute and Late CNS Risks. All of the research and technology questions associated with this risk have priority level one for Mars missions and priority level 3 levels for ISS and 30d lunar missions. The questions addressed by the NSCOR project are 29a (functional changes), 29b (late degeneration), 29c (Alzheimer predisposition), 29e (late degeneration), 29g (cell and tissue mechanisms of damage), 29h (different cell populations including stem cells) and 29i (biomarkers). Also, a NASA workshop on CNS radiobiology identified supporting questions to assist in designing experiments to elucidate CNS effects in response to radiation exposure. These questions include: 1) What is the latency to CNS injury, 2) What cell types are involved in CNS injury (neurons, stem cells, glia)? and 3) What vascular changes occur in response to exposure. We have used this framework to focus our investigation on the effects of charged particles on the CNS. Specifically we will quantify cellular and functional changes in the hippocampus following high energy protons (main SPE constituent) and Fe-56 particles . We will use male rodents with sufficient statistical samples to address inter-individual variation and will address workshop questions 1-3 by examining the responses of different constituent cell types and the latency of damage. NSCOR subproject A quantifies different cell populations (29b, 29e, 29g, 29h); subproject B addresses functional changes and non-invasive techniques for identifying biomarkers (29a, 29i); and subproject C targets late degenerative changes with an Alzheimer model and the role of inflammation (29b, 29c, 29e, 29g and 29i). Our strategy is therefore aligned with the programmatic goals set forth by NASA and its advisory groups.

Technical Progress

Irradiations. Most of the animals scheduled for iron irradiation at the Brookhaven National Laboratory have now been treated and are aging to their appropriate harvest dates. Additional groups of transgenic animals are scheduled for irradiation in the October 2006 run. Essentially all hypothesis B animals have been irradiated with both iron ions and protons. Proton irradiations of animals for stereological analysis under hypothesis A will now be started so that subjects can be harvested on a continuous schedule during project years 3 and 4.

Core Activities. All tissue processing, stereological and immunohistochemical methods have been continuously improved and standard protocols have been established. Adjustments to several immunofluorescence protocols were made to compensate for the limited stability of certain antibodies and to correct for image quality when tomato lectin is used to label the lumens of microvessels. Vascular corrosion casting training by Thomas Krucker is scheduled for early September after which time processing at LLU and shipment to Paul Scherrer Institute will commence. The central database is operating stably using the Linux operating system and the set of servers has been moved to a more permanent location. Mr. Brad Karain, who is the administrator, will be leaving for graduate school in the fall and his replacement is being sought. Electrophysiology technician Tyra Lamp has also left for a new position and her replacement is being sought. It is anticipated that over the next 12 months the MRI facility currently operated by the Department of Radiation Medicine will become jointly operated with the Department of Radiology. Associate Director Andre Obenaus will continue to oversee operations. Plans are in the works for a project review to be held in northern California this fall.

Hypothesis A Activities. Stereological analysis of iron irradiated mice has been completed for animals up to 12 months of age. The systematic sections cut from each brain slice containing compartments of interest were stained to identify the capillaries, nuclei of endothelial cells as well as neurons and glia. Microvessel endothelial cell population numbers as well as vessel lengths in CA1 and dentate gyrus (DG) regions of the hippocampus 6 and 12 months following 56Fe were measured but no significant reduction of endothelial cell density and capillary density was noted at the 6 month time point. However, the stereological methods were shown to be sensitive and demonstrated that there were highly significant differences in microvessel length density as well as endothelial number between dentate gyrus and hilus or CA1 region of the hippocampus. Previous work with rat retinal vasculature suggests that late degenerative changes in microvasculature will first become manifest at about 15 months. With respect to microglia there appears to be an effect of 4 Gy iron irradiation but the results are not yet statistically significant. Certain methodological setbacks required re-imaging and re-counting of a number of sections because the IBA1 antibody appears to be unstable and the tissues must be sectioned, labeled, and imaged with very little margin for delay. We have since modified the protocols appropriately. Most of the sections have already been re-imaged and we are in the process of counting.

For analysis of neurogenesis, tissues have been processed from 57 mice representing doses of 0 to 4 Gy of iron and follow-up times of 1 and 6 months. Many of the sections have been stained and analyzed. Additional tissues from 28 mice that had received varying doses 2 months previously have also been sectioned and stained. Analyses of the latter animals is underway. Based on the analyses carried out to date, it is clear that 56Fe irradiation induces a significant and persistent decrease in the production of new neurons within the dentate gyrus. Furthermore, the reductions appear to be associated with alterations in the microenvironment, including increased indications of inflammation and oxidative stress.

For assessment of DNA damage and repair, hippocampal tissues were isolated from animals exposed to a range of iron doses at 8 weeks after irradiation and processed for transgene mutant frequencies (MF). The spontaneous MF in the hippocampus is consistent with our historical whole brain values. We observed a trend of increasing MF with increasing radiation dose up to 2 Gy. Analyses of characteristics of mutants obtained from the 8 week samples using restriction fragment length polymorphism ( RFLP) are in progress. Pooled results from >50 mutants from the 2 Gy treatment group suggest that > 70% of the mutants contain either point mutations or <1 kb deletions. These results suggest that only cells containing small changes in the genome continue to survive in tissues at 8 weeks post irradiation. Heavily damaged cells containing large genomic damage are most probably eliminated from the surviving cell population at this late time.

Hypothesis B activities. Electrophysiology: Recordings were obtained at 1-3 mo after irradiation. Animals underwent standard in vitro electrophysiological preparation, yielding 400 um thick hippocampal sections. Extracellular recordings were performed in the CA1 dendritic region. After electrophysiological characterization, long term potentiation (LTP) was induced by tetanic stimulation with recordings for 90 mins post LTP induction. Hippocampal LTP was readily induced in 0 Gy animals but this contrasts with head-only irradiated animals at 1 mo after irradiation which had dose-dependent decrements in PTP. Similarly, LTP in 1 mo post-irradiation was decreased at 4 Gy. However, in 3 mo post irradiation animals, there was no significant change between 0 Gy and treated animals. These results demonstrate altered hippocampal plasticity at 1 mo after 56Fe head-only irradiation. At 3 mo these changes appeared to normalize but underlying pathology may lead to other altered hippocampal electrophysiological characteristics.

Magnetic Resonance Imaging : After the transfer of technology involved in small animal diffusion tensor imaging (DTI), the challenge of incorporating non-compatible data format of the MR data in between Washington University and LLU has been addressed. Several custom made computer programs have been created to accept data generated from LLU’s system to construct the quantitative DTI maps. Briefly, the required information embedded in the parameter set of Bruker MRI (the MR system used in LLU) data set was extracted and fed to programs written in Matlab for DTI map calculation. A water phantom was tested by acquiring a DTI data set according to our specifications. The homogeneously distributed image intensity in the raw data suggests a negligible level of artifact in the data set. So, we have established the necessary software to handle the DTI data set for analysis. To ensure the quality of DTI on mouse brain, a Washington University animal holder design has been fabricated and tested at LLU and a custom designed surface coil has been adopted for improved image quality.

Cerebral metabolic rate of oxygen utilization (CMRO2) is a critical physiological parameter for insights into tissue viability during acute cerebral ischemia and we hypothesize that it will reveal vasculature changes following irradiation. This parameter has been studied using modified T2 images acquired after middle cerebral artery occlusion (MCAO) in a rat model. The methods are being adapted at University of North Carolina first to mice then to the Brucker MRI system in use at LLU. A number of software components have now been developed for this purpose and implementation on the LLU Brucker is anticipated in the near future.

Hypothesis C Activities. Electrophysiology: In immunologically challenged mice (lipopolysaccharide injection) the most significant finding was the inhibitory effect of radiation on excitability of CA1 neurons. This was detected as a decrease of the amplitude of population spike evoked at maximal stimulation intensity. The effect was apparent 3 month after radiation, became very significant at the dose of 4 Gy six months after radiation and at 12 months after radiation the effect was clearly dose-dependent. This indicates detrimental effect of radiation predominantly on dendro-somatic coupling function that is responsible for generation of the action potential (functional output of CA1 neurons). In APP23 transgenic mice we measured changes of selected electrophysiological parameters (excitability, synaptic efficacy, short-term and long-term synaptic plasticity) at iron radiation doses of 0, 1, 2 and 4 Gy. In 9 month old mice we were able to detect changes in synaptic efficacy following 1Gy exposures. The decrease was even more prominent at 4 Gy. Such changes were not observed at the age of 2 and 6 months and at the age of 14 months the decrease was less significant due to the reduction of synaptic efficacy in control, non-irradiated mice, presumably caused by aging. The change in synaptic efficacy may reflect changes in synaptic density and dendritic arborization of CA1 neurons due to radiation exposure.

Vascular corrosion casting of transgenic mice will begin in September 2006. The delays were due to switch in location of the APP23 animal colony and the revision of contractual arrangements for synchrotron x-ray tomography.

17th Annual NASA Space Radiation Health Investigators’ Workshop proceedings, June 2006. , Jun-2006

ISMRM 14th Scientific Meeting, Jan 2006. , Jan-2006

17th Annual NASA Space Radiation Health Investigators’ Workshop proceedings, June 2006. , Jun-2006

52nd Annual Meeting of Radiation Research Society proceedings, Oct 2005. , Oct-2005

17th Annual NASA Space Radiation Health Investigators’ Workshop proceedings, June 2006. , Jun-2006

17th Annual NASA Space Radiation Health Investigators’ Workshop proceedings, June 2006. , Jun-2006

17th Annual NASA Space Radiation Health Investigators’ Workshop, June 2006. , Jun-2006

Society for Neuroscience, Washington 2005, poster 503.9. , Nov-2005

52nd Annual Meeting of the Radiation Research Society proceedings, Oct 2005. , Oct-2005

Foremost in the Critical Path Roadmap system for radiation effects on the nervous system is Risk # 39 - Damage to the Central Nervous System from Radiation Exposure. The highest priority questions identified with this risk are: 10.01 Are the biological effects of protons above 10 MeV sufficiently similar to photons that photon data can be used for their consequences? 10.04 Are their differences in response to particles with similar LET, but with different atomic numbers and energies? 10.05 Are there unique biological effects associated with HZE’s? 10.07 How can animal and cell experiments be done and data best be used to extrapolate to the human risk from space radiation? 10.10 What are the risks from SPE’s and what is their impact on operations, EVAs and surface exploration? and 10.12 What are the effects of age, gender, and inter-individual diversity? CPR radiation risks 38, 40, 41 and 42 pose similar questions. A recent NASA workshop (10) identified supporting questions to assist in designing experiments to elucidate central nervous system (CNS) effects in response to radiation exposure. These questions include: 1) What is the latency of CNS injury? 2) What cell types are involved in CNS injury (axons, neurons, glia)? and 3) What vascular changes occur in response to exposure. We have used this framework to focus our investigation on the effects of charged particles on the CNS. Specifically we will quantify cellular and functional changes in the hippocampus following high energy proton (main constituent of SPE’s; Q’s 10.01, 10.10) and 56Fe particle exposure (HZE’s of different LET; Q’s 10.04, 10.05). We will use male rodents with sufficient statistical samples to address inter-individual variation (Q 10.12) and will address workshop questions 1 through 3 by examining the responses of the different constituent cells of brain tissue and the latency of damage. Our strategy is therefore aligned with the programmatic goals set forth by NASA and its advisory groups.

Core activities: Irradiation techniques have been standardized with the use of stereotactic positioners for animals held under anesthesia in a system that pre-aligns mouse brains with collimator openings. Beam transport simulations and dosimetric measurements have been made with 1000 MeV/n and 600 MeV/n iron beams to characterize the irradiation conditions and a complementary collimator system for use with proton beams continues to be optimized and characterized. The system is comprised of an anesthesia/stereotactic holder for isoflurane anesthesia, quick disconnect gas connections and a three-part collimator . Personnel associated with all three hypotheses are now trained on the use of this system and are qualified users at the NSRL facility. In addition, four persons, Roman Vlkolinsky, Ph.D., Scripps Research Institute electrophysiologist, Shu-Wei Sun, Ph.D. Washington University MRI physicist, and LLU technical staff members Tamako Jones and Ella Lloyd have received advanced radiobiology training at BNL as part of the 2004 NASA Space Radiation Summer School Substantial progress has been made in establishing a database system at Loma Linda for archiving and cross referencing of all data sets generated by the NSCOR. Mr. Brad Karain has led this information technology effort which included setting up of a multi-terabyte server system, MySQL software-based database development using the Linux operating system. Data in formats ranging from MRI images to electrophysiological recordings to histological photographs and physical dosimetry are now entered into the database and indexed to individual animal subjects and time-dose cohorts.

Hypothesis A Activities. Immunohistochemical methods are now standardized for identification of specific cell types. Confocal microscope images of hippocampal sections labeled for atrocytes and microvessel-associated pericytes are being assembled into sequential stacks by stereology software developed for use on the Biorad MRC2400 confocal microscope system. Bromodeoxyuridine incorporated into the newly-synthesized DNA of dividing neuronal stem cells in subgranular layer of hippocampus can be visualized by anti-BrdUTP antibodies to quantified newly- born neurons cells and assess the ability of the irradiated brain to repopulate damaged regions. The cells later migrate and integrate into the hippocampal circuitry. Tissue from certain time and dose points for irradiated lacZ-gene containing transgenic mice has been archived for DNA extraction and mutation analysis. Plans for continued irradiation of mice with iron ions and protons are in place for the second year of the project.

Hypothesis B Activities. Three electrophysiology rigs with common architecture and components have been set up for hippocampal slice recordings at LLU, Scripps Research Institute, and U.C.L.A. Input-output curves, long term potentiation and other physiological measurements on control and irradiated animals have been acquired for a limited number of subjects as the cohort ages. Preliminary analysis of recordings show dose-dependent changes in intrinsic nerve electrical properties and in long term potentiation (simple learning paradigm) reflecting capacity for synaptic remodeling following iron ion irradiation. Similar observations were obtained from Hypothesis C using transgenic and LPS-treated animals. Standard magnetization sequences (T1 and T2 relaxations) and diffusion tensor images have been obtained for mouse brains using the Loma Linda Brucker MRI systems (4.7 T and 11.7T). White matter tracts (which constrain fluid diffusion along their longitudinal dimension) can be mapped in three dimensions. Magnetization sequences were developed at Washington University in St. Louis for implemented on the Loma Linda Brucker MRI system so that animal shipping and handling would be minimized. Data acquisition has begun to examine possible demyelination or restructuring of fiber tracts in irradiated animals. Similarly, oxygen extraction fraction sequences have been improved by the U.N.C. group and transformed for use on the Loma Linda Brucker MRI systems.

Hypothesis C Activities. Corrosion casts of control and APP23 mouse brains have been obtained by T. Krucker et al. enabling us to map the three dimensional topology of blood vessels. Regions of brain with and without infarcts caused by Amyloid plaque accumulation can be mapped for infarct frequency vs. age and image processing improvements now permit hippocampal areas to be specifically highlighted. 120 APP23 animals have been irradiated as of 4/7/05 and 138 C57Bl for LPS immune stressor treatments. We have found that the APP23 animals are somewhat fragile and do not ship well, leading to some losses following BNL irradiations. Most of these animals are scheduled for electrophysiological measurements with the majority of corrosion cast animals scheduled for years 3 and 4. Induction of inflammatory response by lipopolysaccharide (LPS) treatment has been visualized by glial cell activation marker CD68 in immunofluorescent preparations using antigen IBA1 to identify glial cells. The strategy of identifying activation markers has been substituted for initial plans to measure soluble cytokines. Electrophysiological measurements following LPS treatments show that LPS-induced inhibition of long term potentiation occurs as expected. Irradiated animals additionally show decreased dendritic excitability and reduced synaptic efficacy in the short term. Data suggest that these changes may recover after 4 months. As can be seen from the examples above, the various methods are in place for carrying out the experimental plan and the NSCOR team is actively acquiring data from irradiated animals which must be maintained for up to 2 years post-irradiation before tissues are harvested for analysis. Irradiations will continue to populate the necessary time-dose cohorts for analysis of tissue structure and performance from 30 days to 2 years post exposure. Arrangements for the next round of iron irradiations are in place for October 2005 at NSRL.

Presentations

Nelson, G.; Archambeau, J.; Chang, P.; Fike, J.; Krucker, T.; Lin, W.; Mao, X.; Obenaus, A.; Pecaut, M.; Song, S.; Spigelman, I. "Progressive alterations of central nervous system structure and function are caused by charged particle radiation. " 16th Annual NASA Space Radiation Investigators’ Workshop. Port Jefferson, N.Y., May 15-18, 2005.

Nelson, G.; Archambeau, J.; Chang, P.; Fike, J.; Krucker, T.; Lin, W.; Mao, X.; Obenaus, A.; Pecaut, M.; Song, S.; Spigelman, I. "Progressive alterations of central nervous system structure and function are caused by charged particle radiation. " Bioastronautics Investigators’ Workshop. Galveston, TX. Jan. 10-12, 2005.

Nelson, G.; Archambeau, J.; Chang, P.; Fike, J.; Krucker, T.; Lin, W.; Mao, X.; Obenaus, A.; Pecaut, M.; Song, S.; Spigelman, I. "Progressive alterations of central nervous system structure and function are caused by charged particle radiation. " Presentation F2.2-0012-04. 35th COSPAR Assembly, Paris. Jul. 19, 2004.

Nelson, G.; Archambeau, J.; Chang, P.; Fike, J.; Krucker, T.; Lin, W.; Mao, X.; Obenaus, A.; Pecaut, M.; Song, S.; Spigelman, I. "Progressive alterations of central nervous system structure and function are caused by charged particle radiation. " . 3rd International Workshop on Space Radiation Research. Port Jefferson, N.Y. May 16-20, 2004.

Obenaus, A.; Robbins, M.; Kendall, E.; Smith, A.; Nelson, G. "Diffusion weighted imaging and magnetic resonance spectroscopy of the central nervous system after high LET radiation: temporal evolution." 16th Annual NASA Space Radiation Investigators' Workshop. Port Jefferson, N.Y., May 15-18, 2005 May-2005