Pharmacologic Manipulation of Complement Receptor 3 Prevents Dendritic Spine Loss and Cognitive Impairment After Acute Cranial Radiation.

PURPOSE: Cranial irradiation induces healthy tissue damage that can lead to neurocognitive complications, negatively affecting patient quality of life. One damage indicator associated with cognitive impairment is loss of neuronal spine density. We previously demonstrated that irradiation-mediated spine loss is microglial complement receptor 3 (CR3) and sex dependent. We hypothesized that these changes are associated with late-delayed cognitive deficits and amenable to pharmacologic intervention. METHODS AND MATERIALS: Our model of cranial irradiation (acute, 10 Gy gamma) used male and female CR3-wild type and CR3-deficient Thy-1 YFP mice of C57BL/6 background. Forty-five days after irradiation and behavioral testing, we quantified spine density and markers of microglial reactivity in the hippocampal dentate gyrus. In a separate experiment, male Thy-1 YFP C57BL/6 mice were treated with leukadherin-1, a modulator of CR3 function. RESULTS: We found that male mice demonstrate irradiation-mediated spine loss and cognitive deficits but that female and CR3 knockout mice do not. These changes were associated with greater reactivity of microglia in male mice. Pharmacologic manipulation of CR3 with LA1 prevented spine loss and cognitive deficits in irradiated male mice. CONCLUSIONS: This work improves our understanding of irradiation-mediated mechanisms and sex dependent responses and may help identify novel therapeutics to reduce irradiation-induced cognitive decline and improve patient quality of life.

Gas6 induces inflammation and reduces plaque burden but worsens behavior in a sex-dependent manner in the APP/PS1 model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease is the leading cause of dementia worldwide. TAM receptor tyrosine kinases (Tyro3, Axl, MerTK) are known for their role in engagement of phagocytosis and modulation of inflammation, and recent evidence suggests a complex relationship between Axl, Mer, and microglial phagocytosis of amyloid plaques in AD. Gas6, the primary CNS TAM ligand, reduces neuroinflammation and improves outcomes in murine models of CNS disease. Therefore, we hypothesized that AAV-mediated overexpression of Gas6 would alleviate plaque pathology, reduce neuroinflammation, and improve behavior in the APP/PS1 model of Alzheimer's disease. METHODS: Adeno-associated viral vectors were used to overexpress Gas6 in the APP/PS1 model of Alzheimer's disease. Nine-month-old male and female APP/PS1 and nontransgenic littermates received bilateral stereotactic hippocampal injections of AAV-Gas6 or AAV-control, which expresses a non-functional Gas6 protein. One month after injections, mice underwent a battery of behavioral tasks to assess cognitive function and brains were processed for immunohistochemical and transcriptional analyses. RESULTS: Gas6 overexpression reduced plaque burden in male APP/PS1 mice. However, contrary to our hypothesis, Gas6 increased pro-inflammatory microglial gene expression and worsened contextual fear conditioning compared to control-treated mice. Gas6 overexpression appeared to have no effect on phagocytic mechanisms in vitro or in vivo as measured by CD68 immunohistochemistry, microglial methoxy-04 uptake, and primary microglial uptake of fluorescent fibrillar amyloid beta. CONCLUSION: Our data describes a triad of worsened behavior, reduced plaque number, and an increase in proinflammatory signaling in a sex-specific manner. While Gas6 has historically induced anti-inflammatory signatures in the peripheral nervous system, our data suggest an alternative, proinflammatory role in the context of Alzheimer's disease pathology.

Cranial irradiation acutely and persistently impairs injury-induced microglial proliferation.

Microglia, the resident immune cells of the central nervous system (CNS), play multiple roles in maintaining CNS homeostasis and mediating tissue repair, including proliferating in response to brain injury and disease. Cranial irradiation (CI), used for the treatment of brain tumors, has a long-lasting anti-proliferative effect on a number of cell types in the brain, including oligodendrocyte progenitor and neural progenitor cells; however, the effect of CI on CNS-resident microglial proliferation is not well characterized. Using a sterile cortical needle stab injury model in mice, we found that the ability of CNS-resident microglia to proliferate in response to injury was impaired by prior CI, in a dose-dependent manner, and was nearly abolished by a 20 â€‹Gy dose. Similarly, in a metastatic tumor model, prior CI (20 â€‹Gy) reduced microglial proliferation in response to tumor growth. The effect of irradiation was long-lasting; 20 â€‹Gy CI 6 months prior to stab injury significantly impaired microglial proliferation. We also investigated how stab and/or irradiation impacted levels of P2Y12R, CD68, CSF1, IL-34 and CSF1R, factors involved in the brain's normal response to injury. P2Y12R, CD68, CSF1, and IL-34 expression were altered by stab similarly in irradiated mice and controls; however, CSF1R was differentially affected. qRT-PCR and flow cytometry analyses demonstrated that CI reduced overall Csf1r mRNA levels and microglial specific CSF1R protein expression, respectively. Interestingly, Csf1r mRNA levels increased after injury in unirradiated controls; however, Csf1r levels were persistently decreased in irradiated mice, and did not increase in response to stab. Together, our data demonstrate that CI leads to a significant and lasting impairment of microglial proliferation, possibly through a CSF1R-mediated mechanism.

Space radiation does not alter amyloid or tau pathology in the 3xTg mouse model of Alzheimer's disease.

Space radiation is comprised of highly charged ions (HZE particles) and protons that are able to pass through matter and cause radiation-induced injury, including neuronal damage and degeneration, glial activation, and oxidative stress. Previous work demonstrated a worsening of Alzheimer's disease pathology in the APP/PS1 transgenic mouse model, however effects of space radiation on tau pathology have not been studied. To determine whether tau pathology is altered by HZE particle or proton irradiation, we exposed 3xTg mice, which acquire both amyloid plaque and tau pathology with age, to iron, silicon, or solar particle event (SPE) irradiation at 9 months of age and evaluated behavior and brain pathology at 16 months of age. We found no differences in performance in fear conditioning and novel object recognition tasks between groups of mice exposed to sham, iron (10 and 100 cGy), silicon (10 and 100 cGy), or solar particle event radiation (200 cGy), though female mice had higher freezing responses than males. 200 cGy SPE irradiated female mice had fewer plaques than sham-irradiated females but had no differences in tau pathology. Overall, females had worse amyloid and tau pathology at 16 months of age and demonstrated a reduced neuroinflammatory gene expression response to radiation. These findings uncover differences between mouse models following radiation injury and corroborate prior reports of sex differences within the 3xTg mouse model.

IL-1ß-driven amyloid plaque clearance is associated with an expansion of transcriptionally reprogrammed microglia.

BACKGROUND: Neuroinflammation is thought to contribute to the pathogenesis of Alzheimer's disease (AD), yet numerous studies have demonstrated a beneficial role for neuroinflammation in amyloid plaque clearance. We have previously shown that sustained expression of IL-1ß in the hippocampus of APP/PS1 mice decreases amyloid plaque burden independent of recruited CCR2+ myeloid cells, suggesting resident microglia as the main phagocytic effectors of IL-1ß-induced plaque clearance. To date, however, the mechanisms of IL-1ß-induced plaque clearance remain poorly understood. METHODS: To determine whether microglia are involved in IL-1ß-induced plaque clearance, APP/PS1 mice induced to express mature human IL-1ß in the hippocampus via adenoviral transduction were treated with the Aß fluorescent probe methoxy-X04 (MX04) and microglial internalization of fibrillar Aß (fAß) was analyzed by flow cytometry and immunohistochemistry. To assess microglial proliferation, APP/PS1 mice transduced with IL-1ß or control were injected intraperitoneally with BrdU and hippocampal tissue was analyzed by flow cytometry. RNAseq analysis was conducted on microglia FACS sorted from the hippocampus of control or IL-1ß-treated APP/PS1 mice. These microglia were also sorted based on MX04 labeling (MX04+ and MX04- microglia). RESULTS: Resident microglia (CD45loCD11b+) constituted > 70% of the MX04+ cells in both Phe- and IL-1ß-treated conditions, and < 15% of MX04+ cells were recruited myeloid cells (CD45hiCD11b+). However, IL-1ß treatment did not augment the percentage of MX04+ microglia nor the quantity of fAß internalized by individual microglia. Instead, IL-1ß increased the total number of MX04+ microglia in the hippocampus due to IL-1ß-induced proliferation. In addition, transcriptomic analyses revealed that IL-1ß treatment was associated with large-scale changes in the expression of genes related to immune responses, proliferation, and cytokine signaling. CONCLUSIONS: These studies show that IL-1ß overexpression early in amyloid pathogenesis induces a change in the microglial gene expression profile and an expansion of microglial cells that facilitates Aß plaque clearance.

Cranial irradiation mediated spine loss is sex-specific and complement receptor-3 dependent in male mice.

Cranial irradiation is the main therapeutic treatment for primary and metastatic malignancies in the brain. However, cranial radiation therapy produces long-term impairment in memory, information processing, and attention that contribute to a decline in quality of life. The hippocampal neural network is fundamental for proper storage and retrieval of episodic and spatial memories, suggesting that hippocampal signaling dysfunction could be responsible for the progressive memory deficits observed following irradiation. Previous rodent studies demonstrated that irradiation induces significant loss in dendritic spine number, alters spine morphology, and is associated with behavioral task deficits. Additionally, the literature suggests a common mechanism in which synaptic elimination via microglial-mediated phagocytosis is complement dependent and associated with cognitive impairment in aging as well as disease. We demonstrate sexual dimorphisms in irradiation-mediated alterations of microglia activation markers and dendritic spine density. Further, we find that the significant dendritic spine loss observed in male mice following irradiation is microglia complement receptor 3 (CR3)-dependent. By identifying sex-dependent cellular and molecular factors underlying irradiation-mediated spine loss, therapies can be developed to counteract irradiation-induced cognitive decline and improve patient quality of life.

Fractionation enhances acute oligodendrocyte progenitor cell radiation sensitivity and leads to long term depletion.

Ionizing radiation (IR) is commonly used to treat central nervous system (CNS) cancers and metastases. While IR promotes remission, frequent side effects including impaired cognition and white matter loss occur following treatment. Fractionation is used to minimize these CNS late side effects, as it reduces IR effects in differentiated normal tissue, but not rapidly proliferating normal or tumor tissue. However, side effects occur even with the use of fractionated paradigms. Oligodendrocyte progenitor cells (OPCs) are a proliferative population within the CNS affected by radiation. We hypothesized that fractionated radiation would lead to OPC loss, which could contribute to the delayed white matter loss seen after radiation exposure. We found that fractionated IR induced a greater early loss of OPCs than an equivalent single dose exposure. Furthermore, OPC recovery was impaired following fractionated IR. Finally, reduced OPC differentiation and mature oligodendrocyte numbers occurred in single dose and fractionated IR paradigms. This work demonstrates that fractionation does not spare normal brain tissue and, importantly, highlights the sensitivity of OPCs to fractionated IR, suggesting that fractionated schedules may promote white matter dysfunction, a point that should be considered in radiotherapy.

Fractionation Spares Mice From Radiation-Induced Reductions in Weight Gain But Does Not Prevent Late Oligodendrocyte Lineage Side Effects.

PURPOSE: To determine the late effects of fractionated versus single-dose cranial radiation on murine white matter. METHODS AND MATERIALS: Mice were exposed to 0 Gy, 6 × 6 Gy, or 1 × 20 Gy cranial irradiation at 10 to 12 weeks of age. Endpoints were assessed through 18 months from exposure using immunohistochemistry, electron microscopy, and electrophysiology. RESULTS: Weight gain was temporarily reduced after irradiation; greater loss was seen after single versus fractionated doses. Oligodendrocyte progenitor cells were reduced early and late after both single and fractionated irradiation. Both protocols also increased myelin g-ratio, reduced the number of nodes of Ranvier, and promoted a shift in the proportion of small, unmyelinated versus large, myelinated axon fibers. CONCLUSIONS: Fractionation does not adequately spare normal white matter from late radiation side effects.

Brain radiation injury leads to a dose- and time-dependent recruitment of peripheral myeloid cells that depends on CCR2 signaling.

BACKGROUND: Cranial radiotherapy is used to treat tumors of the central nervous system (CNS), as well as non-neoplastic conditions such as arterio-venous malformations; however, its use is limited by the tolerance of adjacent normal CNS tissue, which can lead to devastating long-term sequelae for patients. Despite decades of research, the underlying mechanisms by which radiation induces CNS tissue injury remain unclear. Neuroinflammation and immune cell infiltration are a recognized component of the CNS radiation response; however, the extent and mechanisms by which bone marrow-derived (BMD) immune cells participate in late radiation injury is unknown. Thus, we set out to better characterize the response and tested the hypothesis that C-C chemokine receptor type 2 (CCR2) signaling was required for myeloid cell recruitment following brain irradiation. METHODS: We used young adult C57BL/6 male bone marrow chimeric mice created with donor mice that constitutively express enhanced green fluorescent protein (eGFP). The head was shielded to avoid brain radiation exposure during chimera construction. Radiation dose and time response studies were conducted in wild-type chimeras, and additional experiments were performed with chimeras created using donor marrow from CCR2 deficient, eGFP-expressing mice. Infiltrating eGFP+ cells were identified and quantified using immunofluorescent microscopy. RESULTS: Brain irradiation resulted in a dose- and time-dependent infiltration of BMD immune cells (predominately myeloid) that began at 1 month and persisted until 6 months following ≥15 Gy brain irradiation. Infiltration was limited to areas that were directly exposed to radiation. CCR2 signaling loss resulted in decreased numbers of infiltrating cells at 6 months that appeared to be restricted to cells also expressing major histocompatibility complex class II molecules. CONCLUSIONS: The potential roles played by infiltrating immune cells are of current importance due to increasing interest in immunotherapeutic approaches for cancer treatment and a growing clinical interest in survivorship and quality of life issues. Our findings demonstrate that injury from brain radiation facilitates a dose- and time-dependent recruitment of BMD cells that persists for at least 6 months and, in the case of myeloid cells, is dependent on CCR2 signaling.

Arginase 1+ microglia reduce Aß plaque deposition during IL-1ß-dependent neuroinflammation.

BACKGROUND: Neuroinflammation has long been considered a driver of Alzheimer's disease progression. However, experiments developed to explore the interaction between neuroinflammation and Alzheimer's disease (AD) pathology showed a surprising reduction in amyloid beta (Aß) plaque deposition. We sought to understand this unexpected outcome by examining microglia phenotypes during chronic neuroinflammation. METHODS: Using an adeno-associated virus vector carrying hIL-1ß cDNA, inflammation was induced in one hippocampus of 8-month-old amyloid precursor protein (APP)/PS1 mice for 4 weeks, while the other hemisphere received control injections. Bone marrow chimeras and staining analysis were used to identify the origins and types of immune cells present during sustained inflammation. Arginase 1 (Arg1) and inducible nitric oxide synthase (iNOS) immunoreactivity were used as markers of alternatively activated and classically activated cells, respectively, and changes in cellular uptake of Aß by Arg1+ or iNOS+ microglia was demonstrated by confocal microscopy. To determine if an anti-inflammatory phenotype was present during neuroinflammation, RNA was extracted on flow-sorted microglia and rt-PCR was performed. Interleukin-4 injection was used to induce alternatively activated cells, whereas a minipump and intrahippocampal cannula was used to deliver an interleukin (IL)-4Rα antibody to block the induction of Arg1+ cells in the setting of sustained IL-1ß expression. RESULTS: We observed a robust upregulation of centrally derived Arg1+ microglia present only in the inflamed hemisphere. Furthermore, in the inflamed hemisphere, greater numbers of Arg1+ microglia contained Aß when compared to iNOS+ microglia. RNA isolated from flow-sorted microglia from the inflamed hemisphere demonstrated elevation of mRNA species consistent with alternative activation as well as neuroprotective genes such as BDNF and IGF1. To explore if Arg1+ microglia mediated plaque reduction, we induced Arg1+ microglia with IL-4 and observed significant plaque clearance. Moreover, when we reduced Arg1+ microglia induction in the context of neuroinflammation using an anti-IL-4Rα antibody delivered via intrahippocampal cannula, we observed a clear correlation between numbers of Arg1+ microglia and plaque reduction. CONCLUSIONS: Together, these findings suggest that Arg1+ microglia are involved in Aß plaque reduction during sustained, IL-1ß-dependent neuroinflammation, opening up possible new avenues for immunomodulatory therapy of AD.

Central nervous system effects of whole-body proton irradiation.

Space missions beyond the protection of Earth's magnetosphere expose astronauts to an environment that contains ionizing proton radiation. The hazards that proton radiation pose to normal tissues, such as the central nervous system (CNS), are not fully understood, although it has been shown that proton radiation affects the neurogenic environment, killing neural precursors and altering behavior. To determine the time and dose-response characteristics of the CNS to whole-body proton irradiation, C57BL/6J mice were exposed to 1 GeV/n proton radiation at doses of 0-200 cGy and behavioral, physiological and immunohistochemical end points were analyzed over a range of time points (48 h-12 months) postirradiation. These experiments revealed that proton radiation exposure leads to: 1. an acute decrease in cell division within the dentate gyrus of the hippocampus, with significant differences detected at doses as low as 10 cGy; 2. a persistent effect on proliferation in the subgranular zone, at 1 month postirradiation; 3. a decrease in neurogenesis at doses as low as 50 cGy, at 3 months postirradiation; and 4. a decrease in hippocampal ICAM-1 immunoreactivity at doses as low as 10 cGy, at 1 month postirradiation. The data presented contribute to our understanding of biological responses to whole-body proton radiation and may help reduce uncertainty in the assessment of health risks to astronauts. These findings may also be relevant to clinical proton beam therapy.

Interleukin-1ß mediated amyloid plaque clearance is independent of CCR2 signaling in the APP/PS1 mouse model of Alzheimer's disease.

Neuroinflammation is a key component of Alzheimer's disease (AD) pathogenesis. Particularly, the proinflammatory cytokine interleukin-1 beta (IL-1ß) is upregulated in human AD and believed to promote amyloid plaque deposition. However, studies from our laboratory have shown that chronic IL-1ß overexpression in the APPswe/PSEN1dE9 (APP/PS1) mouse model of AD ameliorates amyloid pathology, increases plaque-associated microglia, and induces recruitment of peripheral immune cells to the brain parenchyma. To investigate the contribution of CCR2 signaling in IL-1ß-mediated amyloid plaque clearance, seven month-old APP/PS1/CCR2(-/-) mice were intrahippocampally transduced with a recombinant adeno-associated virus serotype 2 containing the cleaved form of human IL-1ß (rAAV2-IL-1ß). Four weeks after rAAV2-IL-1ß transduction, we found significant reductions in 6E10 and Congo red staining of amyloid plaques that was confirmed by decreased levels of insoluble Aß1-42 and Aß1-40 in the inflamed hippocampus. Bone marrow chimeric studies confirmed the presence of infiltrating immune cells following IL-1ß overexpression and revealed that dramatic reduction of CCR2(+) peripheral mononuclear cell recruitment to the inflamed hippocampus did not prevent the ability of IL-1ß to induce amyloid plaque clearance. These results suggest that infiltrating CCR2(+) monocytes do not contribute to IL-1ß-mediated amyloid plaque clearance.

Thermal injury lowers the threshold for radiation-induced neuroinflammation and cognitive dysfunction.

The consequences of radiation exposure alone are relatively well understood, but in the wake of events such as the World War II nuclear detonations and accidents such as Chernobyl, other critical factors have emerged that can substantially affect patient outcome. For example, ~70% of radiation victims from Hiroshima and Nagasaki received some sort of additional traumatic injury, the most common being thermal burn. Animal data has shown that the addition of thermal insult to radiation results in increased morbidity and mortality. To explore possible synergism between thermal injury and radiation on brain, C57BL/6J female mice were exposed to either 0 or 5 Gy whole-body gamma irradiation. Irradiation was immediately followed by a 10% total-body surface area full thickness thermal burn. Mice were sacrificed 6 h, 1 week or 6 month post-injury and brains and plasma were harvested for histology, mRNA analysis and cytokine ELISA. Plasma analysis revealed that combined injury synergistically upregulates IL-6 at acute time points. Additionally, at 6 h, combined injury resulted in a greater upregulation of the vascular marker, ICAM-1 and TNF-α mRNA. Enhanced activation of glial cells was also observed by CD68 and Iba1 immunohistochemistry at all time points. Additionally, doublecortin staining at 6 months showed reduced neurogenesis in all injury conditions. Finally, using a novel object recognition test, we observed that only mice with combined injury had significant learning and memory deficits. These results demonstrate that thermal injury lowers the threshold for radiation-induced neuroinflammation and long-term cognitive dysfunction.

Chronic neuron- and age-selective down-regulation of TNF receptor expression in triple-transgenic Alzheimer disease mice leads to significant modulation of amyloid- and Tau-related pathologies.

Neuroinflammation, through production of proinflammatory molecules and activated glial cells, is implicated in Alzheimer's disease (AD) pathogenesis. One such proinflammatory mediator is tumor necrosis factor α (TNF-α), a multifunctional cytokine produced in excess and associated with amyloid ß-driven inflammation and cognitive decline. Long-term global inhibition of TNF receptor type I (TNF-RI) and TNF-RII signaling without cell or stage specificity in triple-transgenic AD mice exacerbates hallmark amyloid and neurofibrillary tangle pathology. These observations revealed that long-term pan anti-TNF-α inhibition accelerates disease, cautions against long-term use of anti-TNF-α therapeutics for AD, and urges more selective regulation of TNF signaling. We used adeno-associated virus vector-delivered siRNAs to selectively knock down neuronal TNF-R signaling. We demonstrate divergent roles for neuronal TNF-RI and TNF-RII where loss of opposing TNF-RII leads to TNF-RI-mediated exacerbation of amyloid ß and Tau pathology in aged triple-transgenic AD mice. Dampening of TNF-RII or TNF-RI+RII leads to a stage-independent increase in Iba-1-positive microglial staining, implying that neuronal TNF-RII may act nonautonomously on the microglial cell population. These results reveal that TNF-R signaling is complex, and it is unlikely that all cells and both receptors will respond positively to broad anti-TNF-α treatments at various stages of disease. In aggregate, these data further support the development of cell-, stage-, and/or receptor-specific anti-TNF-α therapeutics for AD.

Sustained IL-1ß expression impairs adult hippocampal neurogenesis independent of IL-1 signaling in nestin+ neural precursor cells.

Alterations in adult hippocampal neurogenesis have been observed in numerous neurological diseases that contain a neuroinflammatory component. Interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine that contributes to neuroinflammation in many CNS disorders. Our previous results reveal a severe reduction in adult hippocampal neurogenesis due to focal and chronic expression of IL-1ß in a transgenic mouse model, IL-1ß(XAT), that evokes a complex neuroinflammatory response. Other investigators have shown that IL-1ß can bind directly to neural precursors to cause cell cycle arrest in vitro. In order to observe if IL-1 signaling is necessary in vivo, we conditionally knocked out MyD88, an adapter protein essential for IL-1 signaling, in nestin(+) neural precursor cells (NPCs) in the presence of IL-1ß-dependent inflammation. Our results show that conditional knockout of MyD88 does not prevent IL-1ß-induced reduction in neuroblasts using a genetic fate mapping model. Interestingly, MyD88 deficiency in nestin(+) NPCs causes an increase in the number of astrocytes in the presence of IL-1ß, suggesting that MyD88-dependent signaling is important in limiting astroglial differentiation due to inflammation. MyD88 deficiency does not alter the fate of NPCs in the absence of inflammation. Furthermore, the inflammatory milieu due to IL-1ß is not affected by the absence of MyD88 in nestin(+) NPCs. These results show that sustained IL-1ß causes a reduction in adult hippocampal neurogenesis that is independent of MyD88-dependent signaling in nestin(+) NPCs, suggesting an indirect negative effect of IL-1ß on neurogenesis.

X-ray microbeam irradiation of the contusion-injured rat spinal cord temporarily improves hind-limb function.

Spinal cord injury is a devastating condition with no effective treatment. The physiological processes that impede recovery include potentially detrimental immune responses and the production of reactive astrocytes. Previous work suggested that radiation treatment might be beneficial in spinal cord injury, although the method carries risk of radiation-induced damage. To overcome this obstacle we used arrays of parallel, synchrotron-generated X-ray microbeams (230 µm with 150 µm gaps between them) to irradiate an established model of rat spinal cord contusion injury. This technique is known to have a remarkable sparing effect in tissue, including the central nervous system. Injury was induced in adult female Long-Evans rats at the level of the thoracic vertebrae T9-T10 using 25 mm rod drop on an NYU Impactor. Microbeam irradiation was given to groups of 6-8 rats each, at either Day 10 (50 or 60 Gy in-beam entrance doses) or Day 14 (50, 60 or 70 Gy). The control group was comprised of two subgroups: one studied three months before the irradiation experiment (n = 9) and one at the time of the irradiations (n = 7). Hind-limb function was blindly scored with the Basso, Beattie and Bresnahan (BBB) rating scale on a nearly weekly basis. The scores for the rats irradiated at Day 14 post-injury, when using t test with 7-day data-averaging time bins, showed statistically significant improvement at 28-42 days post-injury (P < 0.038). H&E staining, tissue volume measurements and immunohistochemistry at day ≈ 110 post-injury did not reveal obvious differences between the irradiated and nonirradiated injured rats. The same microbeam irradiation of normal rats at 70 Gy in-beam entrance dose caused no behavioral deficits and no histological effects other than minor microglia activation at 110 days. Functional improvement in the 14-day irradiated group might be due to a reduction in populations of immune cells and/or reactive astrocytes, while the Day 10/Day 14 differences may indicate time-sensitive changes in these cells and their populations. With optimizations, including those of the irradiation time(s), microbeam pattern, dose, and perhaps concomitant treatments such as immunological intervention this method may ultimately reach clinical use.

Galactic cosmic radiation leads to cognitive impairment and increased aß plaque accumulation in a mouse model of Alzheimer's disease.

Galactic Cosmic Radiation consisting of high-energy, high-charged (HZE) particles poses a significant threat to future astronauts in deep space. Aside from cancer, concerns have been raised about late degenerative risks, including effects on the brain. In this study we examined the effects of (56)Fe particle irradiation in an APP/PS1 mouse model of Alzheimer's disease (AD). We demonstrated 6 months after exposure to 10 and 100 cGy (56)Fe radiation at 1 GeV/µ, that APP/PS1 mice show decreased cognitive abilities measured by contextual fear conditioning and novel object recognition tests. Furthermore, in male mice we saw acceleration of Aß plaque pathology using Congo red and 6E10 staining, which was further confirmed by ELISA measures of Aß isoforms. Increases were not due to higher levels of amyloid precursor protein (APP) or increased cleavage as measured by levels of the ß C-terminal fragment of APP. Additionally, we saw no change in microglial activation levels judging by CD68 and Iba-1 immunoreactivities in and around Aß plaques or insulin degrading enzyme, which has been shown to degrade Aß. However, immunohistochemical analysis of ICAM-1 showed evidence of endothelial activation after 100 cGy irradiation in male mice, suggesting possible alterations in Aß trafficking through the blood brain barrier as a possible cause of plaque increase. Overall, our results show for the first time that HZE particle radiation can increase Aß plaque pathology in an APP/PS1 mouse model of AD.

Cranial irradiation leads to acute and persistent neuroinflammation with delayed increases in T-cell infiltration and CD11c expression in C57BL/6 mouse brain.

Radiotherapy is commonly employed to treat cancers of the head and neck and is increasingly used to treat other central nervous system (CNS) disorders. Exceeding the radiation tolerance of normal CNS tissues can result in sequelae contributing to patient morbidity and mortality. Animal studies and clinical experience suggest that neuroinflammation plays a role in the etiology of these effects; however, detailed characterization of this response has been lacking. Therefore, a dose-time investigation of the neuroinflammatory response after single-dose cranial irradiation was performed using C57BL/6 mice. Consistent with previous reports, cranial irradiation resulted in multiphasic inflammatory changes exemplified by increased transcript levels of inflammatory cytokines, along with glial and endothelial cell activation. Cranial irradiation also resulted in acute infiltration of neutrophils and a delayed increase in T cells, MHC II-positive cells, and CD11c-positive cells seen first at 1 month with doses ≥ 15 Gy. CD11c-positive cells were found almost exclusively in white matter and expressed MHC II, suggesting a "mature" dendritic cell phenotype that remained elevated out to 1 year postirradiation. Our results indicate that cranial irradiation leads to persistent neuroinflammatory changes in the C57BL/6 mouse brain that includes unique immunomodulatory cell populations.

Regulation of prostaglandin E2 synthesis after brain irradiation.

PURPOSE: A local tissue reaction, termed neuroinflammation, occurs after irradiation of brain tissue. Previous work suggested that cyclooxygenase (COX)-2 activity was important for changes in gene expression associated with neuroinflammation as well as increased prostaglandin E2 (PGE2) levels seen after radiation treatment. METHODS AND MATERIALS: To begin to determine the contributions of other enzymes involved in PGE2 production, we examined protein levels of COX-1 and COX-2 as well as 2 PGE synthases (membrane and cytosolic PGES) 4 h after 35 Gy single dose irradiation to the brains of C3HeN mice. We also evaluated the effects of specific COX inhibitors on PGE2 production and PGES expression. RESULTS: As expected, COX-2 expression increased after radiation exposure. Brain irradiation also increased tissue protein levels for both PGES isoforms. Specific COX-2 inhibition with NS398 lowered brain PGE2 levels by about 60%. Surprisingly, COX-1 inhibition with SC560 completely prevented the elevation of PGE2 seen after irradiation. Interestingly, NS398 reduced the membrane-associated PGES isoform, whereas SC560 treatment lowered cytosolic isoform levels below those seen in unirradiated controls. CONCLUSIONS: Taken together, these data indicate that both cyclooxygenases contribute to PGE2 production in irradiated brain and reveal dependence of PGES isoforms expression on specific cyclooxygenase activities.