Cranial irradiation disrupts homeostatic microglial dynamic behavior.

Cranial irradiation causes cognitive deficits that are in part mediated by microglia, the resident immune cells of the brain. Microglia are highly reactive, exhibiting changes in shape and morphology depending on the function they are performing. Additionally, microglia processes make dynamic, physical contacts with different components of their environment to monitor the functional state of the brain and promote plasticity. Though evidence suggests radiation perturbs homeostatic microglia functions, it is unknown how cranial irradiation impacts the dynamic behavior of microglia over time. Here, we paired in vivo two-photon microscopy with a transgenic mouse model that labels cortical microglia to follow these cells and determine how they change over time in cranial irradiated mice and their control littermates. We show that a single dose of 10 Gy cranial irradiation disrupts homeostatic cortical microglia dynamics during a 1-month time course. We found a lasting loss of microglial cells following cranial irradiation, coupled with a modest dysregulation of microglial soma displacement at earlier timepoints. The homogeneous distribution of microglia was maintained, suggesting microglia rearrange themselves to account for cell loss and maintain territorial organization following cranial irradiation. Furthermore, we found cranial irradiation reduced microglia coverage of the parenchyma and their surveillance capacity, without overtly changing morphology. Our results demonstrate that a single dose of radiation can induce changes in microglial behavior and function that could influence neurological health. These results set the foundation for future work examining how cranial irradiation impacts complex cellular dynamics in the brain which could contribute to the manifestation of cognitive deficits.

Role of inflammation in a rat model of radiation retinopathy.

Radiation retinopathy (RR) is a major side effect of ocular tumor treatment by plaque brachytherapy or proton beam therapy. RR manifests as delayed and progressive microvasculopathy, ischemia and macular edema, ultimately leading to vision loss, neovascular glaucoma, and, in extreme cases, secondary enucleation. Intravitreal anti-VEGF agents, steroids and laser photocoagulation have limited effects on RR. The role of retinal inflammation and its contribution to the microvascular damage occurring in RR remain incompletely understood. To explore cellular and vascular events after irradiation, we analyzed their time course at 1 week, 1 month and 6 months after rat eyes received 45 Gy X-beam photons. Müller glial cells, astrocytes and microglia were rapidly activated, and these markers of retinal inflammation persisted for 6 months after irradiation. This was accompanied by early cell death in the outer retina, which persisted at later time points, leading to retinal thinning. A delayed loss of small retinal capillaries and retinal hypoxia were observed after 6 months, indicating inner blood‒retinal barrier (BRB) alteration but without cell death in the inner retina. Moreover, activated microglial cells invaded the entire retina and surrounded retinal vessels, suggesting the role of inflammation in vascular alteration and in retinal cell death. Radiation also triggered early and persistent invasion of the retinal pigment epithelium by microglia and macrophages, contributing to outer BRB disruption. This study highlights the role of progressive and long-lasting inflammatory mechanisms in RR development and demonstrates the relevance of this rat model to investigate human pathology.

NLRP3 promotes radiation-induced brain injury by regulating microglial pyroptosis.

PURPOSE: Radiation-induced brain injury, one of the side effects of cranial radiotherapy in tumour patients, usually results in durable and serious cognitive disorders. Microglia are important innate immune-effector cells in the central nervous system. However, the interaction between microglia and neurons in radiation-induced brain injury remains uncharacterised. METHODS AND MATERIALS: We established a microglia-neuron indirect co-culture model to assess the interaction between them. Microglia exposed to radiation were examined for pyroptosis using lactate dehydrogenase (LDH) release, Annexin V/PI staining, SYTOX staining and western blot. The role of nucleotide-binding oligomerisation domain-like receptor family pyrin domain containing 3 (NLRP3) was investigated in microglia exposed to radiation and in mouse radiation brain injury model through siRNA or inhibitor. Mini-mental state examination and cytokines in blood were performed in 23 patients who had experienced cranial irradiation. RESULTS: Microglia exerted neurotoxic features after radiation in the co-culture model. NLRP3 was up-regulated in microglia exposed to radiation, and then caspase-1 was activated. Thus, the gasdermin D protein was cleaved, and it triggered pyroptosis in microglia, which released inflammatory cytokines. Meanwhile, treatment with siRNA NLRP3 in vitro and NLRP3 inhibitor in vivo attenuated the damaged neuron cell and cognitive impairment, respectively. What is more, we found that the patients after radiation with higher IL-6 were observed to have a decreased MMSE score. CONCLUSIONS: These findings indicate that radiation-induced pyroptosis in microglia may promote radiation-induced brain injury via the secretion of neurotoxic cytokines. NLRP3 was evaluated as an important mediator in radiation-induced pyroptosis and a promising therapeutic target for radiation-induced brain injury.

Galactic Cosmic Ray Particle Exposure Does Not Increase Protein Levels of Inflammation or Oxidative Stress Markers in Rat Microglial Cells In Vitro.

Astronauts on exploratory missions will be exposed to galactic cosmic rays (GCR), which can induce neuroinflammation and oxidative stress (OS) and may increase the risk of neurodegenerative disease. As key regulators of inflammation and OS in the CNS, microglial cells may be involved in GCR-induced deficits, and therefore could be a target for neuroprotection. This study assessed the effects of exposure to helium (4He) and iron (56Fe) particles on inflammation and OS in microglia in vitro, to establish a model for testing countermeasure efficacy. Rat microglia were exposed to a single dose of 20 cGy (300 MeV/n) 4He or 2 Gy 56Fe (600 MeV/n), while the control cells were not exposed (0 cGy). Immediately following irradiation, fresh media was applied to the cells, and biomarkers of inflammation (cyclooxygenase-2 [COX-2], nitric oxide synthase [iNOS], phosphorylated IκB-α [pIκB-α], tumor necrosis factor-α [TNFα], and nitrite [NO2-]) and OS (NADPH oxidase [NOX2]) were assessed 24 h later using standard immunochemical techniques. Results showed that radiation did not increase levels of NO2- or protein levels of COX-2, iNOS, pIκB-α, TNFα, or NOX2 compared to non-irradiated control conditions in microglial cells (p > 0.05). Therefore, microglia in isolation may not be the primary cause of neuroinflammation and OS following exposures to helium or iron GCR particles.

Daytime Light Deficiency Leads to Sex- and Brain Region-Specific Neuroinflammatory Responses in a Diurnal Rodent.

Seasonal changes in peripheral inflammation are well documented in both humans and animal models, but seasonal changes in neuroinflammation, especially the impact of seasonal lighting environment on neuroinflammation remain unclear. To address this question, the present study examined the effects of environmental lighting conditions on neuroinflammation in a diurnal rodent model, Nile grass rats (Arvicanthis niloticus). Male and female grass rats were housed in either bright (brLD) or dim (dimLD) light during the day to simulate a summer or winter light condition, respectively. After 4 weeks, microglia markers Iba-1 and CD11b, as well as pro-inflammatory cytokines TNF-α and IL-6, were examined in the anterior cingulate cortex (ACC), basolateral amygdala (BLA), and dorsal hippocampus (dHipp). The results revealed that winter-like dim light during the day leads to indicators of increased neuroinflammation in a brain site- and sex-specific manner. Specifically, relatively few changes in the neuroinflammatory markers were observed in the ACC, while numerous changes were found in the BLA and dHipp. In the BLA, winter-like dimLD resulted in hyper-ramified microglia morphology and increased expression of the pro-inflammatory cytokine IL-6, but only in males. In the dHipp, dimLD led to a higher number and hyper-ramified morphology of microglia as well as increased expression of CD11b and TNF-α, but only in females. Neuroinflammatory state is thus influenced by environmental light, differently in males and females, and could play a role in sex differences in the prevalence and symptoms of psychiatric or neurological disorders that are influenced by season or other environmental light conditions. Diurnal Nile grass rats were housed under bright or dim light during the day for 4 weeks, simulating seasonal fluctuations in daytime lighting environment. Dim light housing resulted in hyper-ramified morphology of microglia (scale bar, 15 µm) and altered expression of pro-inflammatory cytokines (TNF-α) in a sex- and brain region-specific manner.

Behavioral performance and microglial status in mice after moderate dose of proton irradiation.

Cognitive impairment is a remote effect of gamma radiation treatment of malignancies. The major part of the studies on the effect of proton irradiation (a promising alternative in the treatment of radio-resistant tumors and tumors located close to critical organs) on the cognitive abilities of laboratory animals and their relation to morphological changes in the brain is rather contradictory. The aim of this study was to investigate cognitive functions and the dynamics of changes in morphological parameters of hippocampal microglial cells after 7.5 Gy of proton irradiation. Two months after the cranial irradiation, 8- to 9-week-old male SHK mice were tested for total activity, spatial learning, as well as long- and short-term hippocampus-dependent memory. To estimate the morphological parameters of microglia, brain slices of control and irradiated animals each with different time after proton irradiation (24 h, 7 days, 1 month) were stained for microglial marker Iba-1. No changes in behavior or deficits in short-term and long-term hippocampus-dependent memory were found, but an impairment of episodic memory was observed. A change in the morphology of hippocampal microglial cells, which is characteristic of the transition of cells to an activated state, was detected. One day after proton exposure in the brain tissue, a slight decrease in cell density was observed, which was restored to the control level by the 30th day after treatment. The results obtained may be promising with regard to the future use of using high doses of protons per fraction in the irradiation of tumors.

Gamma frequency entrainment attenuates amyloid load and modifies microglia.

Changes in gamma oscillations (20-50 Hz) have been observed in several neurological disorders. However, the relationship between gamma oscillations and cellular pathologies is unclear. Here we show reduced, behaviourally driven gamma oscillations before the onset of plaque formation or cognitive decline in a mouse model of Alzheimer's disease. Optogenetically driving fast-spiking parvalbumin-positive (FS-PV)-interneurons at gamma (40 Hz), but not other frequencies, reduces levels of amyloid-ß (Aß)1-40 and Aß 1-42 isoforms. Gene expression profiling revealed induction of genes associated with morphological transformation of microglia, and histological analysis confirmed increased microglia co-localization with Aß. Subsequently, we designed a non-invasive 40 Hz light-flickering regime that reduced Aß1-40 and Aß1-42 levels in the visual cortex of pre-depositing mice and mitigated plaque load in aged, depositing mice. Our findings uncover a previously unappreciated function of gamma rhythms in recruiting both neuronal and glial responses to attenuate Alzheimer's-disease-associated pathology.

In vivo imaging reveals transient microglia recruitment and functional recovery of photoreceptor signaling after injury.

Microglia respond to damage and microenvironmental changes within the central nervous system by morphologically transforming and migrating to the lesion, but the real-time behavior of populations of these resident immune cells and the neurons they support have seldom been observed simultaneously. Here, we have used in vivo high-resolution optical coherence tomography (OCT) and scanning laser ophthalmoscopy with and without adaptive optics to quantify the 3D distribution and dynamics of microglia in the living retina before and after local damage to photoreceptors. Following photoreceptor injury, microglia migrated both laterally and vertically through the retina over many hours, forming a tight cluster within the area of visible damage that resolved over 2 wk. In vivo OCT optophysiological assessment revealed that the photoreceptors occupying the damaged region lost all light-driven signaling during the period of microglia recruitment. Remarkably, photoreceptors recovered function to near-baseline levels after the microglia had departed the injury locus. These results demonstrate the spatiotemporal dynamics of microglia engagement and restoration of neuronal function during tissue remodeling and highlight the need for mechanistic studies that consider the temporal and structural dynamics of neuron-microglia interactions in vivo.

Photobiomodulation inhibits the activation of neurotoxic microglia and astrocytes by inhibiting Lcn2/JAK2-STAT3 crosstalk after spinal cord injury in male rats.

BACKGROUND: Neurotoxic microglia and astrocytes begin to activate and participate in pathological processes after spinal cord injury (SCI), subsequently causing severe secondary damage and affecting tissue repair. We have previously reported that photobiomodulation (PBM) can promote functional recovery by reducing neuroinflammation after SCI, but little is known about the underlying mechanism. Therefore, we aimed to investigate whether PBM ameliorates neuroinflammation by modulating the activation of microglia and astrocytes after SCI. METHODS: Male Sprague-Dawley rats were randomly divided into three groups: a sham control group, an SCI + vehicle group and an SCI + PBM group. PBM was performed for two consecutive weeks after clip-compression SCI models were established. The activation of neurotoxic microglia and astrocytes, the level of tissue apoptosis, the number of motor neurons and the recovery of motor function were evaluated at different days post-injury (1, 3, 7, 14, and 28 days post-injury, dpi). Lipocalin 2 (Lcn2) and Janus kinase-2 (JAK2)-signal transducer and activator of transcription-3 (STAT3) signaling were regarded as potential targets by which PBM affected neurotoxic microglia and astrocytes. In in vitro experiments, primary microglia and astrocytes were irradiated with PBM and cotreated with cucurbitacin I (a JAK2-STAT3 pathway inhibitor), an adenovirus (shRNA-Lcn2) and recombinant Lcn2 protein. RESULTS: PBM promoted the recovery of motor function, inhibited the activation of neurotoxic microglia and astrocytes, alleviated neuroinflammation and tissue apoptosis, and increased the number of neurons retained after SCI. The upregulation of Lcn2 and the activation of the JAK2-STAT3 pathway after SCI were suppressed by PBM. In vitro experiments also showed that Lcn2 and JAK2-STAT3 were mutually promoted and that PBM interfered with this interaction, inhibiting the activation of microglia and astrocytes. CONCLUSION: Lcn2/JAK2-STAT3 crosstalk is involved in the activation of neurotoxic microglia and astrocytes after SCI, and this process can be suppressed by PBM.

Ultrasound Stimulation Suppresses LPS-Induced Proinflammatory Responses by Regulating NF-κB and CREB Activation in Microglial Cells.

The purpose of this study was to investigate the effects and underlying mechanisms of low-intensity pulsed ultrasound (LIPUS) against lipopolysaccharide (LPS)-induced neuroinflammation. BV-2 microglia subjected to LPS administration (1 µg/mL) were treated with LIPUS stimulation. The levels of inflammatory mediators and brain-derived neurotrophic factor (BDNF) were quantified using the western blot. The results showed that LIPUS stimulation promoted the associated cAMP response element-binding protein (CREB)/BDNF expression in the LPS-treated microglia. Meanwhile, LIPUS treatment effectively suppressed the LPS-induced production of tumor necrosis factor-α, interleukin-1ß, interleukin-6, inducible nitric oxide synthase, and cyclooxygenase-2 in the microglial cells, in addition to inhibiting the LPS-induced expressions of toll-like receptor 4 and myeloid differentiation factor 88, as well as the LPS-induced activation of c-Jun N-terminal kinase and nuclear factor kappa B. Furthermore, LIPUS significantly decreased the Bax/Bcl-2 ratio in the microglia following LPS treatment. Our data indicated that LIPUS attenuated the proinflammatory responses as well as the decline in BDNF in LPS-treated microglia. This study provides a better understanding of how LIPUS stimulation regulates anti-inflammatory actions in microglia, providing further evidence suggesting that such stimulation may be regarded as a novel strategy for the treatment of neuroinflammation.

Short- and Long-Term Study of the Impact of Focal Blue Light-Emitting Diode-Induced Phototoxicity in Adult Albino Rats.

BACKGROUND: In adult rats we study the short- and long-term effects of focal blue light-emitting diode (LED)-induced phototoxicity (LIP) on retinal thickness and Iba-1+ activation. METHODS: The left eyes of previously dark-adapted Sprague Dawley (SD) rats were photoexposed to a blue LED (20 s, 200 lux). In vivo longitudinal monitoring of retinal thickness, fundus images, and optical retinal sections was performed from 1 to 30 days (d) after LIP with SD-OCT. Ex vivo, we analysed the population of S-cone and Iba-1+ cells within a predetermined fixed-size circular area (PCA) centred on the lesion. RESULTS: LIP resulted in a circular focal lesion readily identifiable in vivo by fundus examination, which showed within the PCAs a progressive thinning of the outer retinal layer, and a diminution of the S-cone population to 19% by 30 d. In parallel to S-cone loss, activated Iba-1+ cells delineated the lesioned area and acquired an ameboid morphology with peak expression at 3 d after LIP. Iba-1+ cells adopted a more relaxed-branched morphology at 7 d and by 14-30 d their morphology was fully branched. CONCLUSION: LIP caused a progressive reduction of the outer retina with loss of S cones and a parallel dynamic activation of microglial cells in the lesioned area.

Regulatory coupling between long noncoding RNAs and senescence in irradiated microglia.

BACKGROUND: Microglia have been implicated in the pathogenesis of radiation-induced brain injury (RIBI), which severely influences the quality of life during long-term survival. Recently, irradiated microglia were speculated to present an aging-like phenotype. Long noncoding RNAs (lncRNAs) have been recognized to regulate a wide spectrum of biological processes, including senescence; however, their potential role in irradiated microglia remains largely uncharacterized. METHODS: We used bioinformatics and experimental methods to identify and analyze the senescence phenotype of irradiated microglia. Western blotting, enzyme-linked immunosorbent assays, immunofluorescence, and quantitative real-time reverse transcription-polymerase chain reaction were performed to clarify the relationship between the radiation-induced differentially expressed lncRNAs (RILs) and the distinctive molecular features of senescence in irradiated microglia. RESULTS: We found that the senescence of microglia could be induced using ionizing radiation (IR). A mutual regulation mode existed between RILs and three main features of the senescence phenotype in irradiated microglia: inflammation, the DNA damage response (DDR), and metabolism. Specifically, for inflammation, the expression of two selected RILs (ENSMUST00000190863 and ENSMUST00000130679) was dependent on the major inflammatory signaling pathways of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK). The two RILs modulated the activation of NF-κB/MAPK signaling and subsequent inflammatory cytokine secretion. For the DDR, differential severity of DNA damage altered the expression profiles of RILs. The selected RIL, ENSMUST00000130679, promoted the DDR. For metabolism, blockade of sterol regulatory element-binding protein-mediated lipogenesis attenuated the fold-change of several RILs induced by IR. CONCLUSIONS: Our findings revealed that certain RILs interacted with senescence in irradiated microglia. RILs actively participated in the regulation of senescence features, suggesting that RILs could be promising intervention targets to treat RIBI.

Signaling pathways involved in anti-inflammatory effects of Pulsed Electromagnetic Field in microglial cells.

Literature studies suggest important protective effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on inflammatory pathways affecting joint and cerebral diseases. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. Therefore the aim of this study was to identify the molecular targets of PEMFs anti-neuroinflammatory action. The effects of PEMF exposure in cytokine production by lipopolysaccharide (LPS)-activated N9 microglial cells as well as the pathways involved, including adenylyl cyclase (AC), phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and delta (PKC-δ), p38, ERK1/2, JNK1/2 mitogen activated protein kinases (MAPK), Akt and caspase 1, were investigated. In addition, the ability of PEMFs to modulate ROS generation, cell invasion and phagocytosis, was addressed. PEMFs reduced the LPS-increased production of TNF-α and IL-1ß in N9 cells, through a pathway involving JNK1/2. Furthermore, they decreased the LPS-induced release of IL-6, by a mechanism not dependent on AC, PLC, PKC-ε, PKC-δ, p38, ERK1/2, JNK1/2, Akt and caspase 1. Importantly, a significant effect of PEMFs in the reduction of crucial cell functions specific of microglia like ROS generation, cell invasion and phagocytosis was found. PEMFs inhibit neuroinflammation in N9 cells through a mechanism involving, at least in part, the activation of JNK MAPK signalling pathway and may be relevant to treat a variety of diseases characterized by neuroinflammation.

Low-Dose Ionizing Radiation Modulates Microglia Phenotypes in the Models of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common type of dementia. AD involves major pathologies such as amyloid-ß (Aß) plaques and neurofibrillary tangles in the brain. During the progression of AD, microglia can be polarized from anti-inflammatory M2 to pro-inflammatory M1 phenotype. The activation of triggering receptor expressed on myeloid cells 2 (TREM2) may result in microglia phenotype switching from M1 to M2, which finally attenuated Aß deposition and memory loss in AD. Low-dose ionizing radiation (LDIR) is known to ameliorate Aß pathology and cognitive deficits in AD; however, the therapeutic mechanisms of LDIR against AD-related pathology have been little studied. First, we reconfirm that LDIR (two Gy per fraction for five times)-treated six-month 5XFAD mice exhibited (1) the reduction of Aß deposition, as reflected by thioflavins S staining, and (2) the improvement of cognitive deficits, as revealed by Morris water maze test, compared to sham-exposed 5XFAD mice. To elucidate the mechanisms of LDIR-induced inhibition of Aß accumulation and memory loss in AD, we examined whether LDIR regulates the microglial phenotype through the examination of levels of M1 and M2 cytokines in 5XFAD mice. In addition, we investigated the direct effects of LDIR on lipopolysaccharide (LPS)-induced production and secretion of M1/M2 cytokines in the BV-2 microglial cells. In the LPS- and LDIR-treated BV-2 cells, the M2 phenotypic marker CD206 was significantly increased, compared with LPS- and sham-treated BV-2 cells. Finally, the effect of LDIR on M2 polarization was confirmed by detection of increased expression of TREM2 in LPS-induced BV2 cells. These results suggest that LDIR directly induced phenotype switching from M1 to M2 in the brain with AD. Taken together, our results indicated that LDIR modulates LPS- and Aß-induced neuroinflammation by promoting M2 polarization via TREM2 expression, and has beneficial effects in the AD-related pathology such as Aß deposition and memory loss.

Potential effects of progranulin and granulins against retinal photoreceptor cell degeneration.

Purpose: The authors previously reported that progranulin attenuated retinal degeneration. The present study focused on the role of progranulin and its cleavage products, granulins, in the pathogenesis of photoreceptor degeneration. Methods: Photoreceptor degeneration was induced with excessive exposure of murine photoreceptor cells and the retinas of albino mice to white fluorescent light. Damaged photoreceptor cells and retinas were examined using a cell death assay, western blotting, and immunostaining. Results: Even after proteolytic cleavage, treatment with progranulin or its cleavage products or both exerted protective effects on photoreceptors against light exposure. In the murine retina, the expression levels of granulins and the macrophage and microglia marker Iba-1 were increased at 48 h after light exposure. Additionally, progranulin+ and Iba-1+ double-positive cells had accumulated in the outer nuclear layer, the primary location of photoreceptor cells. Conclusions: These results suggest that progranulin or its cleavage products, granulins, or both may be therapeutic targets for age-related macular degeneration and other neurodegenerative diseases.

Impact of X-irradiation on microglia.

Irradiation is widely used to treat brain tumors, and also to create bone marrow (BM) chimeras. BM chimeras are widely used to dissect functions and origin of microglia and blood-derived mononuclear cells under homeostatic or pathological conditions. This is facilitated by the fact that microglia survive irradiation and are thus regarded radio-resistant. In this study, we tested whether microglia are indeed radio-resistant and looked for potential mechanisms that might explain this phenomenon. We analyzed the radio-resistance of microglia independently of their physiological brain environment compared to other mononuclear cells from spleen and brain after X-irradiation with 7 Gy or 30 Gy. Furthermore, we investigated long-term effects of X-irradiation on microglia using organotypic hippocampal slice cultures (OHSCs). We found a significant higher survival rate of isolated microglia 4 hr after X-irradiation with 30 Gy accompanied by a decreased proliferation rate. Investigations of apoptosis-related genes revealed no regulation of a specific antiapoptotic pathway but ataxia telangiectasia mutated (ATM), a DNA-repair-related gene, was significantly upregulated in isolated microglia 4 hr after 30 Gy. Irradiation of OHSCs with 7 and 30 Gy revealed a highly and significantly decreased cell number, morphological changes and an increase in migration velocity of microglia. Furthermore, cell loss, increased soma size and process length of microglia was also found in BM chimeras irradiated with 9.5 Gy 5 weeks after irradiation. Here, we present new evidence implying that microglia are not a homogeneous population of radio-resistant cells and report on long-term alterations of microglia that survived irradiation.

Female mice are protected from space radiation-induced maladaptive responses.

Interplanetary exploration will be humankind's most ambitious expedition and the journey required to do so, is as intimidating as it is intrepid. One major obstacle for successful deep space travel is the possible negative effects of galactic cosmic radiation (GCR) exposure. Here, we investigate for the first time how combined GCR impacts long-term behavioral and cellular responses in male and female mice. We find that a single exposure to simulated GCR induces long-term cognitive and behavioral deficits only in the male cohorts. GCR exposed male animals have diminished social interaction, increased anxiety-like phenotype and impaired recognition memory. Remarkably, we find that the female cohorts did not display any cognitive or behavioral deficits after GCR exposure. Mechanistically, the maladaptive behavioral responses observed only in the male cohorts correspond with microglia activation and synaptic loss in the hippocampus, a brain region involved in the cognitive domains reported here. Furthermore, we measured reductions in AMPA expressing synaptic terminals in the hippocampus. No changes in any of the molecular markers measured here are observed in the females. Taken together these findings suggest that GCR exposure can regulate microglia activity and alter synaptic architecture, which in turn leads to a range of cognitive alterations in a sex dependent manner. These results identify sex-dependent differences in behavioral and cognitive domains revealing promising cellular and molecular intervention targets to reduce GCR-induced chronic cognitive deficits thereby boosting chances of success for humans in deep space missions such as the upcoming Mars voyage.

Mitigation of radiation myelopathy and reduction of microglial infiltration by Ramipril, ACE inhibitor.

STUDY DESIGN: Experimental study. OBJECTIVES: To evaluate the efficacy of Angiotensin-converting enzyme inhibitor Ramipril, as a mitigator of radiation-induced spinal cord injury. SETTING: Stony Brook University, Stony Brook, NY, USA. METHODS: Total of 22 rats were irradiated with single doses of 23.6-33 Gy at the C4-T2 spinal levels. After irradiation, the rats were randomized to the radiation only control group and the Ramipril-treated (radiation + Ramipril) experimental group. Ramipril 1.5 mg/kg/day was given in the drinking water starting 1 week after radiation through the study duration. RESULTS: All the rats irradiated with 28.5-33 Gy became paralyzed at 125 ± 4 days, whereas no rats became paralyzed after 23.6 Gy. The time to develop paralysis was delayed to 135 ± 4 days in Ramipril-treated group (P < 0.001). H&E and LFB showed microscopic structural restoration and remyelination with Ramipril treatment. VEGF expression was increased in the irradiated spinal cord, and the number of VEGF-positive cells was significantly decreased by Ramipril treatment (P < 0.001). Immunohistochemical stain with Iba-1 showed increased microglial infiltration in the irradiated spinal cords. The number of Iba-1-positive microglia was significantly reduced by Ramipril treatment (P < 0.05). CONCLUSION: Ramipril reduced the rate of paralysis even at the paralysis-inducing radiation doses. It also significantly delayed the onset of paralysis. Neuroinflammation and endothelial cell damage may be the key mediators of radiation injury. Ramipril can be readily translatable to clinical application as a mitigatory of radiotherapeutic toxicity.

Low-dose fractionated irradiation promotes axonal regeneration beyond reactive gliosis and facilitates locomotor function recovery after spinal cord injury in beagle dogs.

Injury to the adult central nervous system (CNS) results in the formation of glial scar tissues. Glial scar-induced failure of regenerative axon pathfinding may limit axon regrowth beyond the lesion site and cause incorrect reinnervation and dystrophic appearance of stalled growth after CNS trauma. Glial scars also upregulate chondroitin sulphate proteoglycans (CSPGs) and expression of proinflammatory factor(s) that form a barrier to axonal regeneration. Therefore, interventions for glial scarring are an attractive strategy for augmenting axonal sprouting and regeneration and overcoming the physical and molecular barriers impeding functional repair. The glial reaction occurs shortly after spinal cord injury (SCI) and can persist for days or weeks with upregulation of cell cycle proteins. In this study, we utilised Beagle dogs to establish a preclinical SCI model and examine the efficacy of low-dose fractionated irradiation (LDI) treatment, which was performed once a day for 14 days (2 Gy per dose, 28 Gy in total). Low-dose fractionated irradiation is a stable method for suppressing cell activation and proliferation through interference in the cell cycle. Our results demonstrated that LDI could reduce astrocyte and microglia activation/proliferation and attenuate CSPGs and IL-1ß expression. Low-dose fractionated irradiation also promoted and provided a pathway for long-distance axon regeneration beyond the lesion site, induced reinnervation of axonal targets and restored locomotor function after SCI in Beagle dogs. Taken together, our findings suggest that LDI would be a promising therapeutic strategy for targeting glial scarring, promoting axon regeneration and facilitating reconstruction of functional circuits after SCI.

Photobiomodulation with 670 nm light ameliorates Müller cell-mediated activation of microglia and macrophages in retinal degeneration.

Müller cells, the supporting cells of the retina, play a key role in responding to retinal stress by releasing chemokines, including CCL2, to recruit microglia and macrophages (MG/MΦ) into the damaged retina. Photobiomodulation (PBM) with 670 nm light has been shown to reduce inflammation in models of retinal degeneration. In this study, we aimed to investigate whether 670 nm light had an effect on Müller cell-initiated inflammation under retinal photo-oxidative damage (PD) in vivo and in vitro. Sprague-Dawley rats were pre-treated with 670 nm light (9J/cm2) once daily over 5 days prior to PD. The expression of inflammatory genes including CCL2 and IL-1ß was analysed in retinas. In vitro, primary Müller cells dissociated from neonatal rat retinas were co-cultured with 661W photoreceptor cells. Co-cultures were exposed to PD, followed by 670 nm light treatment to the Müller cells only, and Müller cell stress and inflammation were assessed. Primary MG/MΦ were incubated with supernatant from the co-cultures, and collected for analysis of inflammatory activation. To further understand the mechanism of 670 nm light, the expression of COX5a and mitochondrial membrane potential (ΔΨm) were measured in Müller cells. Following PD, 670 nm light-treated Müller cells had a reduced inflammatory activation, with lower levels of CCL2, IL-1ß and IL-6. Supernatant from 670 nm light-treated co-cultures reduced activation of primary MG/MΦ, and lowered the expression of pro-inflammatory cytokines, compared to untreated PD controls. Additionally, 670 nm light-treated Müller cells had an increased expression of COX5a and an elevated ΔΨm following PD, suggesting that retrograde signaling plays a role in the effects of 670 nm light on Müller cell gene expression. Our data indicates that 670 nm light reduces Müller cell-mediated retinal inflammation, and offers a potential cellular mechanism for 670 nm light therapy in regulating inflammation associated with retinal degenerations.