Neurobiology of Aging

The anti-inflammatory efficacy of radiation therapy (RT) with single fractions below 1.0 Gy has been demonstrated in Alzheimer’s disease mouse models. As neuroinflammation is also a major pathological feature of Parkinson’s disease (PD), RT may also be effective in PD treatment. Therefore, this study aimed to investigate the anti-inflammatory effect of low-moderate dose RT (LMDRT, 0.6 Gy/single dose, for 5 days) exposure in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 30 mg/kg, intraperitoneally, for 5 consecutive days)-induced PD mouse model. Importantly, LMDRT reduced the levels of glial fibrillary acidic protein and intercellular adhesion molecule-1 (CD54) in the striatum region, which increased following MPTP administration. LMDRT also modulated inflammatory gene expression patterns in the substantia nigra region of the MPTP-treated mice. However, LMDRT had no direct effects on the severe loss of dopaminergic neurons and impaired motor behavior in the rotarod test. These results indicate that LMDRT has anti-inflammatory effects by modulating neuroinflammatory factors, including glial fibrillary acidic protein and intercellular adhesion molecule-1, but showed no behavioral improvements or neuroprotection in the MPTP-induced mouse model of PD. © 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).


Introduction
The prevalence of Parkinson's disease (PD), a neurodegenerative disorder, has doubled over the past 25 years, with more than 8.5 million patients with PD in 2019 (WHO, 2022).Despite gradual increase of patients with PD, the causes of PD are not yet fully understood, and there is currently no cure for PD.Although several therapeutic agents and deep brain stimulation are performed for PD treatment, they simply induce symptom relief and may be associated with postoperative complications and various side effects due to long-term use.Since the United States Food and Drug Administration approval in 1970, Levodopa, a dopamine precursor, has been used as the most potent treatment for patients with PD for more than 50 years (Hauser, 2009;Lewitt, 2008).Despite the therapeutic benefits of Levodopa, chronic Levodopa therapy may be accompanied by diminished efficacy over time and motor complications, including postural abnormalities, motor fluctuations, and dyskinesia (Thanvi and Lo, 2004).
Numerous studies have shown the involvement of neuroinflammation in PD.Indeed, in the postmortem brains of patients with PD, activated microglia and the reactive astrocytes were noted in the substantia nigra pars compacta (SNpc) (McGeer et al., 1988;Miklossy et al., 2006).Activated microglia have also been found in positron emission tomography scans of early patients with PD (Ouchi et al., 2009(Ouchi et al., , 2005)), while elevated proinflammatory cytokines have been found in both cerebrospinal fluid (transforming growth factor β1, interleukin (IL)-1β, and IL-6) and blood (IL-6, tumor necrosis factor, IL-1β, C-reactive protein, IL-10, Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted, and IL-2) of patients with PD (Chen et al., 2018;Qin et al., 2016).Based on this clinical evidence, neuroinflammation has been considered an important pathological feature of PD (Gelders et al., 2018;Hirsch et al., 2003;Hirsch and Hunot, 2009), and many pharmacological approaches targeting neuroinflammation have been attempted for PD treatment.Despite the efficacy of several anti-inflammatory drugs in preclinical studies (Grotemeyer et al., 2022;Mallah et al., 2020), their clinical efficacy remains insignificant (Gagne and Power, 2010;Ren et al., 2018).Therefore, effective and stable treatment of PD also requires a therapeutic strategy targeting neuroinflammation through a nonpharmacological approach.
There is much clinical evidence that radiation therapy (RT) with single fractions below 1.0 Gy has anti-inflammatory effects by modulating inflammatory immune reactions (Calabrese and Calabrese, 2013;Rödel et al., 2012Rödel et al., , 2007)).In Europe, X-rays with a total dose of 3-6 Gy with a single dose of 0.5-1 Gy are already being used for patients with chronic degenerative arthritis and inflammatory diseases, and approximately 90% of the patients have reduced pain after RT (Keller et al., 2013;Ott et al., 2015).Currently, a clinical study is in progress for patients with severe pneumonia with inflammation after coronavirus disease, and it has shown improvements in some patients after RT with a single dose of 0.5-1 Gy (Dunlap et al., 2021).
Interestingly, RT also has therapeutic effects for neurodegenerative disorders such as Alzheimer's disease (AD) and PD.Cuttler et al. reported that low-dose RT with a single dose of < 0.1 Gy exposure through repeated computed tomography scans improved cognitive function and speech in patients with AD and reduced the tremor symptom of a patient with PD (Boyd et al., 2021;Cuttler et al., 2021Cuttler et al., , 2017Cuttler et al., , 2018)).
Recently, exposure to RT with a total dose of 3 Gy with a single dose of 0.6 Gy in an AD mouse model showed anti-inflammatory and therapeutic effects (Yang et al., 2021).Based on this previous study, RT with a single dose of 0.6 Gy has sufficient potential as a new treatment to achieve anti-inflammatory effects on neurodegenerative disorders.Preclinical results show that pretreatment of wholebody irradiation has an antioxidant effect with a single dose of 0.5 Gy and a neuroprotective potential with a single dose of 3.5 Gy in a PD mouse model (Kojima et al., 1999;Liang et al., 2006), but the anti-inflammatory effects of RT in PD have not yet been elucidated.Therefore, the present study aimed to investigate whether the previously reported anti-inflammatory effects of whole-brain lowmoderate dose radiation therapy (LMDRT: total dose of 3 Gy with a single dose of 0.6 Gy) could also be observed in the 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuroinflammation in a PD mouse model.

Animals
Male C57BL/6 mice (aged 9 weeks, 22-25 g; Orient Bio Inc, Gapyeong, Korea) were used in the experiment.The mice were maintained under a 12-hour light/dark cycle (lights on at 8 AM) under specific pathogen-free conditions.All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Korea Institute of Radiological & Medical Sciences (IACUC No. kirams 2021-0054 and kirams 2022-0088).

Experimental procedure
A total of 60 mice were randomly assigned to 4 groups (n = 15 mice in each group): saline, LMDRT, MPTP, and MPTP+LMDRT.Mice were intraperitoneally injected with saline or MPTP (30 mg/kg/d; Sigma-Aldrich, St. Louis, MO, USA) for 5 consecutive days.After allowing time (3 days) to recover their body weight, the mice were treated with LMDRT for 5 days to investigate the therapeutic effects of LMDRT.Two different cohorts of mice were used in all experiments: the first cohort contained 32 mice (n = 8 mice in each group), and the second cohort included 28 mice (n = 7 mice in each group).In the first cohort, at 7 days after the last LMDRT, mice were sacrificed for immunohistochemistry (n = 5 mice in each group).The remaining 3 mice from each group were sacrificed, and then the SNpc and striatum regions of them were used for cytokine array and microarray analyses, respectively.In the second cohort, mice were subjected to a rotarod test that was performed at 3 time points: before MPTP injection (day 0; baseline), before LMDRT (day 7), and 7 days after the last LMDRT (day 18).After the last rotarod test on day 18, 4 mice in each group of them were used in the enzymelinked immunosorbent assay (ELISA) assay ( Fig. 1A).

Rotarod test
The rotarod apparatus (Panlab/Harvard Apparatus, Holliston, MA, USA) was used to test motor balance and coordination.Before the rotarod test, all mice were habituated and pretrained on the rotarod (15 rpm) for 5 minutes over 3 days.On the test day, the speed of rotation was accelerated gradually from 4 to 40 rpm for 300 seconds, and the total time spent on the rotarod was recorded.Each mouse was tested over 3 trials, with 15-minute intervals between trials.

Cell counting and image quantification
Following a previous study with slight modification (Huang et al., 2022), we analyzed all images using ImageJ software (version 1.53a; Bethesda, MD, USA).Briefly, TH-positive neurons in the SNpc were counted in every sixth serial coronal sections throughout the SNpc (a total of 9 sections).Regions of interest (ROIs) of the left and right hemispheres of the SNpc in each brain section were defined using the polygon tool, and the number of TH-positive neuron in ROIs was counted.The total number of TH-positive neurons in the SNpc was estimated as follows: [the number of TH-positive neurons in the left and right sides of the SNpc in 9 sections] × 6 (every sixth section was sampled).The quantification of axonal degeneration of TH-positive staining in the striatum or staining density of GFAP-positive or Iba1positive cells in the SNpc and striatum was determined as a relative ratio compared to the saline value.To maintain a constant threshold for each image and compensate for subtle variability in the immunostaining, 4 sections were randomly selected, and the SNpc-ROI or striatal-ROI was defined in each section.We only counted pixels that were at least 90% brighter than the average level of each image There is no significant difference in body weight between the groups (F 1,24 = 1.274, p = 0.270 for the main effect of MPTP; F 1,24 = 0.001, p = 0.977 for the main effect of LMDRT; F 1,24 = 0.001, p = 0.974 for interaction between MPTP and LMDRT).(D) Rotarod test.The duration of time that the mice spent on the rotating rod is measured.MPTP significantly reduces the latency times on the rotarod, but there is no significant effect of LMDRT (F 1,24 = 5.525, p = 0.027 for the main effect of MPTP; F 1,24 = 0.160, p = 0.693 for the main effect of LMDRT; F 1,24 = 0.125, p = 0.727 for interaction between MPTP and LMDRT).Data are presented as the mean ± standard error of the mean (n = 7 mice in each group).*p < 0.05 indicates a significant difference based on the 2-way repeated measures analysis of variance followed by Tukey's post hoc test.Abbreviations: Gy, Gray; i.p., intraperitoneal; LMDRT, low-moderate dose radiation therapy; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.
after background subtraction and shading correction.All counting and quantitative procedures described above were performed blind without knowledge of the experimental conditions.

Proteome profiler mouse cytokine array
A Proteome Profiler Mouse Cytokine Array (R&D System, Minneapolis, MN, USA) was used to detect 40 mouse cytokines and chemokines.Briefly, anesthetized mice were decapitated, and their brains were rapidly removed.The striatum tissues were dissected and homogenized in ice-cold PBS with protease inhibitors, and Triton X-100 was added to the final concentration of 1%.Tissues were frozen at −70°C overnight and thawed.After centrifugation, the total proteins were quantitated, and the striatum tissue lysate (200 µg) was used for the cytokine array, following the manufacturer's instructions.

Enzyme-linked immunosorbent assay
ELISA was used for the quantitative detection of a single cytokine.Anesthetized mice were transcardially perfused with PBS, and the brains were removed.The striatum tissues were rapidly dissected and homogenized in PBS and stored at −20°C overnight.After centrifugation, homogenates from the striatum were analyzed for the measurement of mouse intercellular adhesion molecule-1 (ICAM-1; CD54) using a specific Mouse ICAM-1/CD54 ELISA kit (R&D System) according to the user manual.

Microarray analysis
Anesthetized mice were decapitated, and their brains were rapidly removed.The SNpc tissues were dissected and collected in RNAlater solution.The total RNA was isolated from the SNpc tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.After measuring the quality and quantity of total RNA, it was used for microarray analysis.The data of microarray analysis were summarized and normalized using the signal space transformation-robust multichip analysis method implemented in Affymetrix Power Tools (Macrogen, Seoul, Korea).The results were exported with gene-level signal space transformationrobust multichip analysis, and then differentially expressed gene (DEG) analysis was performed.Statistical significance of the expression data was determined using the independent t-test and log 2 fold change, in which the null hypothesis was that no difference would exist between the groups.The false discovery rate was controlled by adjusting the p value using the Benjamini-Hochberg algorithm.DEG analysis was performed when the data satisfied the criteria, that is, log 2 fold change > 1.2 and a significant t-test result (p < 0.05).Hierarchical cluster analysis was performed to compare the differences in the gene expression between the groups based on the DEGs using complete linkage and Euclidean distance as a measure of similarity.Gene enrichment and functional annotation analysis for the significant probe list were performed using Gene Ontology (http://geneontology.org).All data analysis and visualization of differentially expressed genes were conducted using R 3.3.2(www.r-project.org).

Statistical analysis
Graphs were generated using GraphPad Prism 9.5 (La Jolla, CA, USA).All statistical analyses were performed using SPSS Statistics 23 (IBM Corp, Armonk, NY, USA).Two-way repeated measures analysis of variance (ANOVA) followed by Tukey's post hoc test was performed to analyze the body weight and rotarod test results.Two-way ANOVA followed by Tukey's post hoc test was also performed to analyze the immunohistochemistry, cytokine assay, and ELISA results.All data are represented as mean ± standard error of the mean, and statistical significance was considered at a p value of < 0.05.

LMDRT had no significant effect on motor impairment in MPTPtreated mice
The rotarod test is widely used to test the behavioral verification of the MPTP-induced PD mouse model by measuring coordinated motor skills of mouse on a rotating rod (Rozas et al., 1998).To investigate the effect of LMDRT on motor impairment in the MPTPinduced PD mouse model, a rotarod test was performed at 3 time points.The entire experimental paradigm is shown in Fig. 1A.A 2way repeated measures ANOVA revealed no significant difference in body weight between the groups (F 1,24 = 1.274, p = 0.270 for the main effect of MPTP; F 1,24 = 0.001, p = 0.977 for the main effect of LMDRT; F 1,24 = 0.001, p = 0.974 for interaction between MPTP and LMDRT; Fig. 1C).During the MPTP treatment period, body weight decreased by approximately 10% but recovered after 3 days.There was no change in body weight during the LMDRT period, indicating the absence of biotoxicity caused by whole-brain irradiation.The 2-way repeated measures ANOVA also revealed that latency times on the rotarod test were significantly reduced after MPTP injections, but there was no significant effect of LMDRT (F 1,24 = 5.525, p = 0.027 for the main effect of MPTP; F 1,24 = 0.160, p = 0.693 for the main effect of LMDRT; F 1,24 = 0.125, p = 0.727 for interaction between MPTP and LMDRT; Fig. 1D).There were no differences in rotarod test performance at all 3 time points in the saline and LMDRT groups.The MPTP group showed significantly reduced performance time after MPTP injections on day 7 (p = 0.028, day 0 vs. 7), and this significance was prolonged on day 18 (p = 0.018, day 0 vs. 18).In the MPTP+LMDRT group, the rotarod test performance time was also significantly reduced after MPTP injections on day 7 (p = 0.046, day 0 vs. 7).However, after LMDRT exposure, the MPTP+LMDRT group showed a slightly increased rotarod test performance time on day 18 (p = 0.166, day 0 vs. 18), but with no significant difference compared with that on day 7 (p = 0.772, day 7 vs. 18; Fig. 1D).These results suggest that LMDRT did not show sufficient efficacy in improving MPTP-induced motor impairment.

LMDRT had no protective effect on dopaminergic neuronal loss in MPTP-treated mice
The MPTP-induced dopaminergic neuronal loss of the nigrostriatal pathway is a major pathological feature of PD (Petroske et al., 2001).To investigate the effects of LMDRT on the loss of dopaminergic neurons caused by MPTP, we performed immunohistochemistry with TH, a dopaminergic neuronal marker in the SNpc (Fig. 2A) and striatum (Fig. 2C) regions.The 2-way ANOVA revealed that MPTP significantly reduced TH-positive immunoreactive neurons in the SNpc, but there was no significant effect of LMDRT (F 1,16 = 62.883, p < 0.0001 for the main effect of MPTP; F 1,16 = 2.450, p = 0.137 for the main effect of LMDRT; F 1,16 = 4.534, p = 0.049 for interaction between MPTP and LMDRT; Fig. 2B).In the striatum region, the 2-way ANOVA revealed that MPTP significantly reduced TH-positive fiber density of dopaminergic neurons, but there was no significant effect of LMDRT (F 1,16 = 48.566,p < 0.0001 for the main effect of MPTP; F 1,16 < 0.0001, p = 0.988 for the main effect of LMDRT; F 1,16 = 1.269, p = 0.277 for interaction between MPTP and LMDRT; Fig. 2D).These results suggest that LMDRT has no neuroprotective effect against MPTP-induced neuronal loss of the nigrostriatal pathway.

LMDRT reduced increasing GFAP expression in the striatum of MPTP-treated mice
Neuroinflammation, which is regulated by the activity of astrocytes, is a major pathological feature of PD (Booth et al., 2017;Li et al., 2019).The expression of GFAP, an astrocyte marker, is increased by MPTP administration and regulated by anti-inflammatory agents in the MPTP-induced PD mouse model (Gong et al., 2019;Zhang et al., 2017).To investigate the anti-inflammatory effect of LMDRT on astrocyte activity, immunohistochemical staining for GFAP was performed in the SNpc (Fig. 3A) and striatum (Fig. 3C) regions.In the SNpc region, there was no significant difference in GFAP expression in all groups (F 1,16 = 0.185, p = 0.673 for the main effect of MPTP; F 1,16 = 0.428, p = 0.522 for the main effect of LMDRT; F 1,16 = 0.052, p = 0.823 for interaction between MPTP and LMDRT; Fig. 3B).In the striatum region, the 2-way ANOVA revealed significant differences in GFAP expression between the groups (F 1,16 = 62.061, p < 0.0001 for the main effect of MPTP; F 1,16 = 7.315, p = 0.016 for the main effect of LMDRT; F 1,16 = 2.518, p = 0.132 for interaction between MPTP and LMDRT; Fig. 3D).The post hoc test results showed that GFAP expression was significantly increased in the MPTP group compared with the saline group (p < 0.0001), whereas the enhanced striatal GFAP expression was significantly reduced in the MPTP+LMDRT group compared with the MPTP group (p = 0.035; Fig. 3D).These results indicate that LMDRT has an antiinflammatory effect by attenuating the increased expression of GFAP in the striatum of MPTP-induced PD mice.

LMDRT reduced proinflammatory cytokine expression in the striatum of MPTP-treated mice
Next, to examine the anti-inflammatory effect of LMDRT, 40 cytokines and chemokines in the striatum were compared between the groups using a cytokine proteome profiler array (Fig. 4A).The 2-way ANOVA showed significant differences between the groups in the relative expression levels of inflammatory cytokines, such as ICAM-1 The density of TH-positive fibers is quantified in the striatum.Each datum was normalized to 100% of the saline value.MPTP significantly reduces the TH-positive fiber density of dopaminergic neurons in the striatum, but there is no significant effect of LMDRT (F 1,16 = 48.566,p < 0.0001 for the main effect of MPTP; F 1,16 < 0.0001, p = 0.988 for the main effect of LMDRT; F 1,16 = 1.269, p = 0.277 for interaction between MPTP and LMDRT).Data are presented as the mean ± standard error of the mean (n = 5 mice in each group).*p < 0.001 and ****p < 0.0001 indicate significant differences based on the 2-way analysis of variance followed by Tukey's post hoc test.Abbreviations: LMDRT, low-moderate dose radiation therapy; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase.

LMDRT changed inflammatory gene expression in the SNpc of MPTP-treated mice
Next, a microarray was performed to examine the inflammatory genes that were significantly changed by LMDRT in the SNpc region.Only 22 of the inflammatory-related genes showed significantly different expression levels with a log 2 fold change > 1.2 (p < 0.05) in the saline versus MPTP group (Fig. 5A) and MPTP versus MPTP +LMDRT group (Fig. 5B).Among the 22 inflammatory genes with significantly different expression levels between the 2 groups, 12 There is no significant difference in GFAP expression in all groups (F 1,16 = 0.185, p = 0.673 for the main effect of MPTP; F 1,16 = 0.428, p = 0.522 for the main effect of LMDRT; F 1,16 = 0.052, p = 0.823 for interaction between MPTP and LMDRT).(C) The photographs are representative images of fluorescent immunohistochemistry of GFAP in the striatum, and (D) the density of GFAP-positive cells is quantified in the striatum.MPTP significantly increases GFAP expression in the MPTP group compared to the saline group, whereas LMDRT significantly reduces GFAP expression in the MPTP +LMDRT group compared with the MPTP group (F 1,16 =62.061, p < 0.0001 for the main effect of MPTP; F 1,16 = 7.315, p = 0.016 for the main effect of LMDRT; F 1,16 = 2.518, p = 0.132 for interaction between MPTP and LMDRT).Each datum was normalized to 100% of the saline value.Data are presented as the mean ± standard error of the mean (n = 5 mice in each group).*p < 0.05, ****p < 0.0001 indicate significant differences based on the 2-way analysis of variance followed by Tukey's post hoc test.Abbreviations: GFAP, glial fibrillary acidic protein; LMDRT, low-moderate dose radiation therapy; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SNpc, substantia nigra pars compacta.
genes were upregulated, and 10 genes were downregulated in the MPTP group compared with the saline group (Fig. 5A).In the MPTP +LMDRT group, 8 genes were upregulated, and 14 genes were downregulated compared with the MPTP group (Fig. 5B).These results suggest that the inflammatory gene expression patterns were changed by LMDRT.

Discussion
Recently, anti-inflammatory agents have been shown to relieve PD symptoms through immune system regulation and neuroprotective effects in a PD mouse model (Ghosh et al., 2012;Maatouk et al., 2019).However, because most clinical studies have shown conflicting results depending on the dose of the agent and the progression of PD, there remain difficulties in clinical application (Di Luca et al., 2022).To overcome the limitations of the pharmacological approach targeting inflammation in PD, we demonstrated that LMDRT (total dose of 3 Gy with a single dose of 0.6 Gy), which is currently used clinically for inflammation treatment, had an antiinflammatory effect in a PD mouse model.
Reactive astrocytes are closely related to neuroinflammation in patients with PD (Miklossy et al., 2006), and they are found in animal models of PD induced by MPTP, 6-hydroxydopamine, and αsynuclein overexpression (Roca et al., 2011).Therefore, to clarify whether LMDRT modulates neuroinflammation by inhibiting reactive astrocytes, the expression of GFAP-positive astrocytes was measured in both SNpc and striatum of MPTP-induced mice.LMDRT significantly reduced the increased expression of GFAP in the striatum of MPTP-induced PD mice; however, this change did not appear in the SNpc (Fig. 3).Interestingly, LMDRT reduced the increased expression of proinflammatory cytokine-related ICAM-1 in the striatum of MPTP-induced PD mice (Fig. 4C).ICAM-1, which is known to be overexpressed in inflammatory responses, is expressed in GFAP-immunoreactive astrocytes (Lee and Benveniste, 1999;Miguel-Hidalgo et al., 2007;Müller, 2019;Rothlein et al., 1986).Miklossy et al. reported the presence of ICAM-1-positive astrocytes in the brains of patients with PD and in the MPTP-induced PD animal model, which were involved in a continuous inflammatory process (Miklossy et al., 2006).This suggests that LMDRT may contribute to suppressing the inflammatory reaction by reducing the increases in GFAP and ICAM-1 expression in the striatum of MPTP-treated mice.Additionally, the revelation that LMDRT regulates ICAM-1-positive astrocytes through double immunostaining will provide a major biomarker for immunomodulation of LMDRT in PD.The anti-inflammatory efficacy of RT can be explained by the radiobiological mechanisms.RT with single fractions below 1.0 Gy reduced inflammation by increasing the anti-inflammatory action of macrophages and reduced the immune response by modulating cytokine, nitric oxide production, and release of reactive oxygen species (ROS) (Calabrese and Calabrese, 2013;Rödel et al., 2012Rödel et al., , 2007)).In particular, ROS promotes neuroinflammation and plays an important role in neuronal damage, which is also closely related to MPTP-induced PD pathology (Haddad, 2002).According to a previous study, the level of GSH (glutathione) as an anti-ROS factor was increased after pretreatment with RT with a single dose of 0.5 Gy in the MPTP-induced PD mouse model (Kojima et al., 1999).Additionally, astrocyte plays an important role in the maintenance of brain homeostasis by interacting with neurons and is also responsible for defending against oxidative stress induced by neuroinflammation (Belanger and Magistretti, 2009).Importantly, in the MPTP-induced PD mouse model, GFAP-positive reactive astrocytes are involved in the production of cytotoxic ROS, which cause dopaminergic neuronal damage (Rizor et al., 2019).Although not revealed in this study, if the relationship between cytotoxic ROS and reduced GFAP-positive astrocytes after LMDRT is revealed, it will help us understand the neuroprotective mechanism of the anti-inflammatory effects of LMDRT.
Despite the anti-inflammatory effects of LMDRT, it did not exert direct effects on motor deficits and severely damaged dopaminergic neurons in the striatal pathway in MPTP-induced PD mice.There was even a tendency for the dopaminergic neurons to decrease in the SNpc after LMDRT exposure in the saline group, but this did not substantially affect other biological results, including the density of TH-positive fibers in the striatum, body weight, and locomotor behavior patterns.The reason why the anti-inflammatory effect of LMDRT was not effective in neuroprotection and overcoming of movement disorders in this study can probably be explained by several limitations.First, microglial activation also plays an important role in regulating neuroinflammation in PD (McGeer et al., 1988), but we were not able to reveal the efficacy of LMDRT because microglial activation did not increase by MPTP in the SNpc and striatum (Supplemental file 1).Previous studies showed that microglial activation progressively increased as an early neurotoxin effect of MPTP injection, but over time, it tends to decrease to the level of the resting state (Huang et al., 2017;Yasuda et al., 2008); it is possible that microglial activation returned to the resting state after 14 days of the last MPTP injection.In addition, since LMDRT reduced increased GFAP in the striatum but not in the SNpc, the anti-inflammatory effect may be rather modest to protect severely damaged dopaminergic neurons.Second, the period was too short to investigate the neuroprotective effect of LMDRT.Moreover, damaged dopaminergic neurons were reversible in the MPTP-induced PD mouse model (Petroske et al., 2001), and we also confirmed that reduced TH-positive neurons in the SNpc recovered at 30 days after MPTP injections.For this reason, the effect of slightly increased the rotarod test performance time in the MPTP+LMDRT group was shown on day 18, but the long-term effect was not tested.
The most meaningful point of our investigation was that we are the first to reveal the anti-inflammatory efficacy of LMDRT in a PD mouse model, providing basic scientific data between PD and LMDRT.However, further studies are needed to elucidate the mechanism of action of LMDRT in reactive astrocytes, which underlies the anti-inflammatory effects of LMDRT in patients with PD.

Conclusions
LMDRT (total dose of 3 Gy with a single dose of 0.6 Gy) had antiinflammatory effects in a PD mouse model by reducing the increased expression of GFAP and ICAM-1 and modulating neuroinflammatory-related gene expression, but it had no neuroprotective effect on severely damaged neuron recovery in the MPTP-induced PD mouse model.Overall, the present results can be used as basic nonclinical data for future clinical applications in patients with PD, suggesting for the first time the anti-inflammatory efficacy of LMDRT in PD mouse model.

Verification
This study has not been published previously elsewhere and it is not under consideration for publication elsewhere.The publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out, and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language, including electronically without the written consent of the copyright-holder.

Disclosure statement
None.

Fig. 1 .
Fig. 1.Experimental design, body weight, and rotarod test.(A) Experimental procedure.Mice were exposed to LMDRT (0.6 Gy/d) for 5 d after intraperitoneal injection of saline or MPTP (30 mg/kg/d) for 5 d.Rotarod tests were performed on day 0 (before MPTP injection), day 7 (before LMDRT exposure), and day 18 (7 d after the last irradiation).At 7 d after the last irradiation, the mice were sacrificed and analyzed.(B) The photograph shows the placement of mice for 60 Co γ-ray irradiation.The yellow dashed line indicates the irradiation area (field size: 30 × 5 cm) for whole-brain irradiation.(C) Body weight.There is no significant difference in body weight between the groups (F 1,24 = 1.274, p = 0.270 for the main effect of MPTP; F 1,24 = 0.001, p = 0.977 for the main effect of LMDRT; F 1,24 = 0.001, p = 0.974 for interaction between MPTP and LMDRT).(D) Rotarod test.The duration of time that the mice spent on the rotating rod is measured.MPTP significantly reduces the latency times on the rotarod, but there is no significant effect of LMDRT (F 1,24 = 5.525, p = 0.027 for the main effect of MPTP; F 1,24 = 0.160, p = 0.693 for the main effect of LMDRT; F 1,24 = 0.125, p = 0.727 for interaction between MPTP and LMDRT).Data are presented as the mean ± standard error of the mean (n = 7 mice in each group).*p < 0.05 indicates a significant difference based on the 2-way repeated measures analysis of variance followed by Tukey's post hoc test.Abbreviations: Gy, Gray; i.p., intraperitoneal; LMDRT, low-moderate dose radiation therapy; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Fig. 2 .
Fig. 2. Effects of LMDRT on MPTP-induced loss of dopaminergic neurons in the SNpc and striatum.(A)The photographs are representative images of immunohistochemical staining of TH in the SNpc and (B) the graph shows that the number of TH-positive cell bodies was counted in the SNpc.MPTP significantly reduces TH-positive immunoreactive neurons in the SNpc, but there is no significant effect of LMDRT (F 1,16 = 62.883, p < 0.0001 for the main effect of MPTP; F 1,16 = 2.450, p = 0.137 for the main effect of LMDRT; F 1,16 = 4.534, p = 0.049 for interaction between MPTP and LMDRT).(C) The photographs are representative images of immunohistochemical staining of TH in the striatum.(D) The density of TH-positive fibers is quantified in the striatum.Each datum was normalized to 100% of the saline value.MPTP significantly reduces the TH-positive fiber density of dopaminergic neurons in the striatum, but there is no significant effect of LMDRT (F 1,16 = 48.566,p < 0.0001 for the main effect of MPTP; F 1,16 < 0.0001, p = 0.988 for the main effect of LMDRT; F 1,16 = 1.269, p = 0.277 for interaction between MPTP and LMDRT).Data are presented as the mean ± standard error of the mean (n = 5 mice in each group).*p < 0.001 and ****p < 0.0001 indicate significant differences based on the 2-way analysis of variance followed by Tukey's post hoc test.Abbreviations: LMDRT, low-moderate dose radiation therapy; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase.

Fig. 3 .
Fig. 3. Effects of LMDRT on GFAP expression in the SNpc and striatum of MPTP-treated mice.(A) These are representative images of fluorescent immunohistochemistry of GFAP in the SNpc, (B) the density of GFAP-positive cells is quantified in the SNpc.Dashed circles indicate the SNpc.There is no significant difference in GFAP expression in all groups (F 1,16 = 0.185, p = 0.673 for the main effect of MPTP; F 1,16 = 0.428, p = 0.522 for the main effect of LMDRT; F 1,16 = 0.052, p = 0.823 for interaction between MPTP and LMDRT).(C) The photographs are representative images of fluorescent immunohistochemistry of GFAP in the striatum, and (D) the density of GFAP-positive cells is quantified in the striatum.MPTP significantly increases GFAP expression in the MPTP group compared to the saline group, whereas LMDRT significantly reduces GFAP expression in the MPTP +LMDRT group compared with the MPTP group (F 1,16 =62.061, p < 0.0001 for the main effect of MPTP; F 1,16 = 7.315, p = 0.016 for the main effect of LMDRT; F 1,16 = 2.518, p = 0.132 for interaction between MPTP and LMDRT).Each datum was normalized to 100% of the saline value.Data are presented as the mean ± standard error of the mean (n = 5 mice in each group).*p < 0.05, ****p < 0.0001 indicate significant differences based on the 2-way analysis of variance followed by Tukey's post hoc test.Abbreviations: GFAP, glial fibrillary acidic protein; LMDRT, low-moderate dose radiation therapy; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SNpc, substantia nigra pars compacta.

Fig. 4 .
Fig. 4. Effects of LMDRT on inflammatory cytokines in the striatum of MPTP-treated mice.(A) Representative image of the proteome profiler array of 40 cytokines in each group (n = 2 mice in each group).(B) The relative mean pixel density (fold change of saline value) of 8 cytokines among 40 cytokines is difference between the groups.(C) Protein concentration of ICAM-1 is measured using ELISA in each group (n = 4 mice in each group).MPTP significantly increases the concentration of ICAM-1 in the MPTP group compared to the saline group, whereas LMDRT significantly reduces the concentration of ICAM-1 in the MPTP+LMDRT group compared with the MPTP group (F 1,12 = 39.591,p < 0.0001 for the main effect of MPTP; F 1,12 = 103.136,p < 0.0001 for the main effect of LMDRT; F 1,12 = 122.639,p < 0.0001 for interaction between MPTP and LMDRT).Data are presented as the mean ± standard error of the mean.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 indicates significant differences based on the 2-way analysis of variance followed by Tukey's post hoc test.Abbreviations: ICAM-1, intercellular adhesion molecule-1; IL, interleukin; LMDRT, low-moderate dose radiation therapy; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; RANTES (CCL5), regulated upon activation normal T cell expressed and presumably secreted; TREM-1, triggering receptor expressed on myeloid cells.

Fig. 5 .
Fig. 5. Effects of LMDRT on inflammatory gene expression in the SNpc of MPTP-treated mice.One-way hierarchical clustering heatmap of the microarray based on the z-score of the normalized value (n = 3 mice in each group).The heat map shows significant differences in inflammatory gene expression along with the data satisfying the criteria, that is, log 2 fold change > 1.2 and a significant t-test result (p < 0.05) between the 2 groups: (A) saline versus MPTP, (B) MPTP versus MPTP+LMDRT.Abbreviations: LMDRT, low-moderate dose radiation therapy; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NF-Kb, nuclear factor kappa-light-chain-enhancer of activated B cell.