All experimental procedures were performed following the guidelines for animal care of the National Institute of Health. The ethical committee approved all Beijing Institute of Technology procedures and the Key Laboratory of Heavy ion Radiation Biology and Medicine of the Chinese Academy of Sciences. In the present study, parallel experiments were performed on the two cohorts of rats such that cohort I was irradiated with ^12C6+ ions and cohort II rats was irradiated with ⁵⁶Fe²⁶⁺ ion for 1, 2, and 3 months to study the impact of radiation on the neurobiology of the striatum. A total of 84 male Sprague Dawley (SD) rats (4 weeks old, weighing 180 ± 10 g) were housed in standard conditions with temperature 22 (±1)°C, humidity, 40–50%, 12 h light and dark cycle with free access to food and water to acclimatise normal conditions. All experimental rats (cohort I, cohort II, and the control group) were given free access to food and water. The control groups were treated like the irradiated experimental group except for irradiation. Feeding conditions remain persistent before and after irradiation.

Cohort I was subdivided into three experimental groups (G1, G2, G3: 15 Gy of ¹²C⁶⁺: 1 month, 2 months, and 3 months, respectively), (n = 6) per group, and three control groups (C4, C5, C6: 0Gy: 3 months), (n = 6) per group. Cohort II was subdivided into four experimental groups (G7, G8, G9: 3.4 Gy of ⁵⁶Fe²⁶⁺: 1, 2, 3 months, respectively, and G10: 8 Gy of ⁵⁶Fe²⁶⁺ for 2 months), (n = 6) per group, and four control groups (C11, C12, C13, C14: 0Gy: 3 months), (n = 6) per group. The Heavy Ion Research Facility in Lanzhou (HIRFL), Institute of Modern Physics, Chinese Academy of Sciences received rats for irradiation. The animals from both cohorts were weighed, anaesthetised intraperitoneally with 5% pentobarbital (70 μg/kg), and fixed to an irradiation machine at HIRFL. The whole brain of cohort 1 rats was irradiated with a single dose of ^12C6+ ions at a distance of 200 cm with an intensity of 0.5 Gy/min for 30 min to achieve 15 Gy total dosage (165 MeV/u primary energy; LET, 30 keV/μm). In cohort II (groups 7–9), animals were irradiated with ⁵⁶Fe²⁶⁺ ion radiations at a distance of 200 cm with an intensity of 0.7 Gy/min for 4.8 min to achieve 3.4 Gy dosage, and group 10 animals were irradiated with ⁵⁶Fe²⁶⁺ ion radiations at a distance of 200 cm with an intensity of 0.7 Gy/min for 11.4 min to achieve 8 Gy dosage (163 MeV/u primary energy, LET, 500–1,000 keV). Control groups of both cohorts were anaesthetised and fixed for the same period without irradiation. All irradiated rats from both cohorts and controls were sacrificed after 1, 2, or 3 months, respectively.

After irradiation, rats of each group from both cohorts were transferred to standard laboratory conditions and rehabilitated for 15 days. Behavioural tests were performed after 15 days to assess depressive-like behaviours. An open-field test was initially performed to determine anxious behaviours (Silverman et al., 2013). Rats were exposed to open fields on the 36th, 76th, and 106th day after irradiation to observe their curiosity levels when exposed to the new environment in an open area or prefer to remain near protective walls. The overall activity of each rat was observed, including total time and distance travelled in the central arena, no movement time, local movements (<10 cm), and average velocity of the moment.

After 24 h of the open field test, a rotarod test was performed to evaluate motor disabilities in rats developed after irradiation, as described previously (Brooks and Dunnett, 2009; Yoshihara et al., 2009). Rats were subjected to the rotarod paradigm on the 38th day, 78th day, and 108th day for three trials per day for 3 successive days with a 300-s accelerating program from 5 to 40 rpm, and abeyance to fall from the rod was measured for analysis. Again, rats were normalised for 24 h, and a sugar preference test was performed to determine the anhedonia level as described earlier (Serchov et al., 2016). Each cohort was subjected to tests on the 41st day, 81st day, and 111th day. Sucrose preference levels between control and irradiated groups were analysed using the formula: Sucrose preference percentage (%) = sucrose intake (ml)/[sucrose intake (ml) + water intake (ml)] × 100%.

After behavioural tests, rats were normalised for a few days and subjected to PET (Position emission tomography) scanning on the 45th day, 85th day, and 115th day. Rats were fasted for 8–12 h without free access to water and then anaesthetised with isoflurane. One unit of ¹⁸F-FDG was injected into anaesthetised rats via tail. After 30 min, rats were administered to the imaging device for brain scanning for 15 min, and changes in glucose metabolism were analysed with SPM software.

Each cohort of rats was weighed, deeply anesthetized with pentobarbital sodium (60 mg/kg of body weight, concentration, 20 mg/mL), and then sacrificed. After perfusion with cold saline solution, the striatum, thymus, and spleen were excised on an ice-cold plate then washed with phosphate buffer solution and stored at −80°C for further experimentation. Additionally, thymus, and spleen were weighed before being preserved at −80°C.

RNA was extracted from the striatum of all experimental groups using Trizol reagent (ThermoFisher Waltham, MA, USA). mRNA was enriched with magnetic beads with oligo (DT). After adding fragmentation buffer, the first cDNA strand was synthesised with random hexamers using mRNA as a template and the second cDNA strand was synthesised by adding buffer, dNTPs, and DNA polymerase I. The cDNA was purified using AMPure XP beads, and the end product was repaired. The A-tail was added and connected to the sequencing connector, and ampere XP beads screened the fragment size. The cDNA library was constructed by PCR enrichment. The Qubit2.0 was used for preliminary quantitative detection. After diluting the library to 1 ng/UL, the insert size of the library was detected by the Agilent 2,100 system, and the effective concentration of the library was quantified by the real-time quantitative PCR detecting system (the effective concentration of the library was more than 2 nM). Transcriptome analysis (RNA-seq) was performed by Illumina HiSeqTM 2000.

The extracted RNA samples were sent to Nuohe Zhiyuan company for high-throughput sequencing and transcriptomics detection. Differently expressed genes in transcriptomic detection were found significant, with a log value greater than 1 or less than −1 and a value of p less than 0.05. Significant differentially expressed genes were analysed using the DAVID 6.8 bioinformatics functional tool¹ (Huang et al., 2009). KEGG pathways (Kyoto Encyclopedia of Gene and Genome) and GO-biological process (Gene Ontology) with FDR < 0.05 were identified and visualised by bubble chart with the ggplot R package.

SH-SY5Y, U87, THP-1, U937, and Jurkat were obtained from the cell centre of Peking Union Medical College. Neuron cell line SH-SY5Y was cultured in DMEM medium and Sijiqing serum, while glioma cell line U87 was cultured in MEM (Minimum Essential Medium) with imported foetal bovine serum (Gibco, Life Technologies, Carlsbad, CA, USA) and antibiotics. For irradiated cell culture, fresh or conditioned MEM supplemented with 10% heat-inactivated foetal bovine serum (Gibco, Life Technologies, Carlsbad, CA, USA), 100 units/mL penicillin, and 100 μg/mL streptomycin (Beijing Solarbio Science & Technology Co., Beijing, China). The culture was maintained at 37°C in a humidified incubator containing 5% CO₂.

For irradiation, SH-SY5Y, U87, and their combination (1:1) were seeded at a density of 1.7 × 105 in T25 cell culture flasks. After 12–24 h of incubation, cells were irradiated horizontally with ¹²C⁶⁺ heavy ions at a dose of 1 Gy, 2 Gy, and 5 Gy (165 MeV/u primary energy; linear energy transfer (LET), 30 KeV/μm; intensity, 0.3–0.5 Gy/min) at HIRFL. After irradiation, the fresh medium was replaced immediately and cultured in 37°C incubators for 24 h, after which a conditioned medium from irradiated cells was used to culture immune cell culture and cell viability assay.

Cell viability was analysed through Cell Titer 96® AQueous One Solution Cell Proliferation Assay as per protocol (Cat. G3581, Promega Corporation, Biotech Co., Ltd.). In brief, 100 μL of conditioned medium was seeded with 6,000 immune cells in each 96-well plate. After 24 h, 20 μL of provided reagents were added to each well, incubated for 2 h at 37°C, and evaluated through a fluorometer at 490 nm. The optical density (OD) was used to analyse cell viability. About six duplications were set.

The transwell migration assay was conducted to analyse the transmigration of THP-1. Twenty-four Transwell inserts with a pore size of 0.4 μm (Corning, USA) were utilised. After irradiation, 500 μL aliquots of the conditioned medium were added (lower chamber), and 105 THP-1 cells in 200 μL of serum-free RPMI-1640 medium were added (upper chambers) in the Transwell inserts. The cells were cultured for 24 h and washed in PBS twice after the medium was discarded. Cells were fixed in formaldehyde (5 min) and stained with crystal violet. Finally, the membrane was analysed using an Olympus-IX71 light microscope.

Cytokine levels in condition medium-irradiated cell culture were determined using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions of Global Biotech Co., Ltd. Shanghai, China (#ABIN367992), and other materials were obtained from the Beijing Hankehengyu Bio-Technology Co., Ltd. Beijing, China.

Results were expressed as mean ± standard deviation. The statistical analyses were performed using unpaired two-tailed student’s t-test and two-way ANOVA using GraphPad PRISM 8.0 software. The value of p <0.05(*), p < 0.01(**), and p < 0.001(***) were considered to show statistical significance.

In the present study, rats from both experimental cohorts were exposed to heavy ion radiation vertically on the backside of the head. The cohort-I groups (G1, G2, and G3) irradiated with 15 Gy of ¹²C⁶⁺ ion, and cohort II groups (G7, G8, and G9) irradiated with 3.4 Gy of ⁵⁶Fe²⁶⁺ ion radiations were analysed after 1 to 3 months, respectively, such that G1 and G7 after 1 month, G2 and G8 after 2 months, and G3 and G9 after 3 months. At the same time, group (G10) from cohort II irradiated with 8 Gy ⁵⁶Fe²⁶⁺ ion radiation was analysed after 2 months. All changes in the irradiated groups were compared with control groups.

Initially, physiological changes induced by heavy ion irradiations on the rats were determined by analysing their body weights after exposure to ¹²C⁶⁺ and ⁵⁶Fe²⁶⁺ ion radiations. Variable physiological changes were observed in both cohorts. Our results showed that all three groups (G1, G2, and G3) of cohort I presented significant weight loss after the 1st, 2nd, and 3rd months than the control groups (Figure 1A). However, cohort II (G7, G8, and G9) presented slow changes in weight loss with ⁵⁶Fe²⁶⁺ ion radiation exposure, such that 3.4 Gy irradiated groups (G7, G8, and G9) did not report significant weight changes till 6 weeks compared to the control group. However, after 8 weeks, 3.4 Gy rats reported significant weight loss (p < 0.01) that remained consistent till the 12th week of the experiment than the control group. The G-10 group (8 Gy) initially reported some weight gain for 4 weeks, but prolonged exposure to 8 Gy of ⁵⁶Fe²⁶⁺ ions also resulted in significant weight loss (p < 0.01) compared to the control group (Figure 1B).

Furthermore, the weight of the spleen and thymus of both cohorts were analysed to determine the impact of irradiation on the immune system. It was observed that the weight of the spleen and thymus was significantly reduced in the cohort I group (G1, G2, and G3) than in the control group, as shown in Figures 1C,D. In contrast, cohort II groups (G7, G8, and G9) showed no significant change in spleen mass after the 1st, 2nd, and 3rd month of irradiation, and thymus weight was increased significantly in the 3rd month compared to control groups (Figures 1E,F), pointing hyperfunctioning of the thymus in response to radiation exposure. However, no significant change in thymus and spleen weight was observed after 2 months in the G10 (8 Gy) irradiated group (Figures 1G,H). Altogether, these findings speculate that ¹²C⁶⁺ irradiation with a high dose (15 Gy) has a more damaging effect, as revealed by the significant loss in the body weights along with the spleen and thymus masses in the G1–G3 groups. However, an increase in the thymus size after 3 months of the ⁵⁶Fe²⁶⁺ small dose (3.4 Gy) might be due to the rebound phenomenon after atrophy caused by radiation exposure, as supported by Moini et al. (2020).

Furthermore, to determine the aftereffects of heavy ion radiation on brain functionality, we behaviourally tested rats after 1, 2, and 3 months of exposure to ¹²C⁶⁺ and ⁵⁶Fe²⁶⁺ ion radiations. Variable behavioural abnormalities were observed in both experimental cohorts. Behavioural tests of both cohorts showed that heavy radiation significantly impacts brain function to impair emotions and trigger long-term cognitive dysfunction. Analysis of sucrose preference tests in both cohorts showed that long-term radiation exposure led to anhedonia, a characteristic feature of depressive behaviour. Up to 2 months, cohort II groups (G7 and G8) did not display significant differences in the sugar and water intake than the control group (Figures 2I,J). However, after the third month of irradiation, cohort II group (G9) showed a significantly lower sugar water intake (p < 0.05), as shown in Figure 2K. In line with these findings, the G10 group irradiated with ⁵⁶Fe²⁶⁺ ion (8 Gy) and G2 group irradiated with ¹²C⁶⁺ (15 Gy) showed a significant loss of interest in sugar water after 2 months than the control group (p < 0.05), as shown in Figure 2J, illustrating that high dose irradiations are more lethal and might promote neuronal and neuropsychological issues, compared to low range dose of ⁵⁶Fe²⁶⁺ (3.4 Gy).

The open field test was performed to analyse the anxious behaviour of both irradiated cohorts I and II. Results have shown that the G7, G8, and G9 groups of cohort II (3.4 Gy) developed significant anxiety levels (p < 0.05) when exposed to a new environment (Figures 2A–D) in all 3 months, showing more standing time towards the wall and crossing a large number of squares. However, the severity of anxiety increases in the 2nd and 3rd months of irradiation (p < 0.05) as illustrated by parameters such as a significantly high number of squares travel time and time facing towards the wall (Figures 2B–D). Moreover, the G10 group (⁵⁶Fe²⁶⁺ ion: 8 Gy) and G2 group (¹²C⁶⁺: 15 Gy) of both cohorts showed that rats develop anxiousness after 2 months of irradiation (Figure 2D), as demonstrated by the crossing of the significantly large number of squares (p < 0.05) compared to the control group, indicating that higher dose and long-term effects of radiations pose deleterious impact on the brain functioning and lead to cognitive issues.

Additionally, rota rod tests are utilised to measure motor coordination ability. Initially, both irradiated cohort’s groups (G7: 3.4 Gy, G10: 8 Gy, and G2:15Gy) showed less physical activity and spent significantly less time on rotating wheels (p < 0.05) than the control group after 1 and 2 months of irradiation (Figures 2E,F,H), indicating that irradiation might affect several brain parts, which leads to coordination disabilities to withstand stress. However, after 3 months of irradiation, the cohort II group, G9 (3.4 Gy), spent more time on rotating wheels than the control group, but no significant difference (p = 0.102) was found between the two groups (Figure 2G). Hence, this peculiarity in behaviour reflects that rats might have developed adaptive behaviour towards this stimulus or can be a false negative result that might be influenced by various factors involved during experimentation.

Next, the intensity of brain damage caused by different ⁵⁶Fe²⁶⁺ and ¹²C⁶⁺ irradiations at multiple conditions was investigated by measuring glucose (¹⁸F-FDG) metabolism in the rat’s brain. Both cohorts were injected with ¹⁸F-FDG, a glucose analogue that does not undergo glycolysis when taken up by brain cells. The glucose utilisation and cell viability of different brain regions of both cohorts were analysed by ¹⁸F-FDG-PET scans. In these results, a medium grey/black area shows sites of glucose metabolism and a lighter colour shows more minor changes in glucose metabolism. In contrast, the white area shows no change in glucose metabolism. The results from cohort I showed that the G1 group, after the 1st month of 15Gy ¹²C⁶⁺ irradiations, presented a significant glucose hypometabolism in the striatum, olfactory sphere, and prefrontal joint cortex region than other parts of the brain displaying glucose hypermetabolism (Figure 3A). However, in G2 group after 2nd month of irradiation, changes in the glucose metabolism increased. Among other brain parts, the amygdala, striatum, and prefrontal cortex presented a significant decline in glucose metabolism (Figure 3A), demonstrating that glucose metabolism was more prominent in the brain’s front side. Final observations obtained from the G3 group after the 3rd month showed that the glucose metabolism rate remained almost the same as in the 2nd month. In contrast, the degree and location of glucose metabolic changes remained slightly altered (Figure 3A). Same brain regions, including the amygdala, striatum, and cortex, displayed glucose hypometabolism. The overall analysis showed that glucose hypometabolism penetrated the front brain regions due to prolonged radiation exposure, especially in the amygdala, striatum, and cortex, reflecting impaired functioning of these particular brain regions, particularly sensory and cognitive processing.

Additionally, the cohort II ¹⁸F-FDG-PET scans showed dynamic glucose metabolism changes at multiple time zones. After 1st month of 3.4 Gy ⁵⁶Fe²⁶⁺ irradiation in the G7 group, various brain regions, including the striatum, cortex, and hippocampus, displayed significant glucose hypometabolism than other parts of the brain compared with the control group (Figures 3B,C). Among these regions, the striatum showed the most considerable glucose hypometabolism. The G8 group of cohort II revealed that glucose hypometabolism increases most significantly in the striatum region compared to the control group, demonstrating that decreased cellular metabolism might result in impaired striatum functioning (Figure 3B). Alternatively, in the G9 group, ¹⁸F-FDG-PET scans show glucose hypermetabolism in the striatum region than the control group (Figures 3B,C). However, glucose metabolism of other brain regions did not show any significant difference with the control group.

Interestingly, the analysis of ¹⁸F-FDG-PET scans of the 8 Gy irradiated group (G10) after 2 months of irradiation has demonstrated glucose hypermetabolism in the hippocampus and striatum region, but most pronounced in the striatum region (Figure 3C). However, this peculiarity in glucose metabolism between 8 Gy and 3.4 Gy ¹⁸F-FDG-PET scans after 2 months in G8 and G10 showed that 8 Gy has deeply penetrated the brain and resulted in significant neuronal damage, like the findings as obtained from the G9 of 3.4 Gy-⁵⁶Fe²⁶⁺ irradiation (Figures 3C,D). Therefore, based on these findings and previous reports (Onorato et al., 2020; Mancini et al., 2021), it is hypothesised that hypermetabolism in the striatum region after prolonged irradiation might result in neuronal damage, which triggers neuroinflammation by activating microglia and astrocytes and leads to more glucose consumption to repair striatal damage.

Interestingly, the abnormal glucose metabolism in the striatum is also linked with our behaviour test results, showing voluntary movements in open field tests and behaviour in rotarod tests. Hence, based on the persistent changes observed in the striatum region of both cohorts as exposed by behaviour tests and ¹⁸F-FDG-PET scans, we selected striatum samples of cohort II (G7, G8, G9, and G10 groups, n = 3 per group) for the transcriptomic analysis to identify the underlying molecular changes involved in striatum damage and repairment at multiple radiation conditions.

According to the transcriptomic analysis of striatum samples, about 2,402 genes were significantly upregulated, including EIF3K, FAM96B, CDKN1A, HDAC9, and ZNF211, at different radiation conditions. In contrast, 3,172 genes were significantly downregulated, including EVI2B, GLRA1, PMFBP1, and PNMAL2, compared to the control (Figure 4C). The top 10 upregulated signalling pathways involved in regulating the striatum of irradiated cohort II are illustrated in Figure 4A. KEGG analysis of differentially expressed genes among these groups shows that neuroinflammatory pathways are highly active along with metabolic pathways. G7 group (3.4 Gy) shows that the mTOR signalling pathway, neuroactive ligand-receptor interaction pathway, Gap junction pathway, GABAergic synapse pathway, protein digestion and absorption pathway, inflammatory mediator regulation of TRP channels pathway, glutamatergic synapse pathway, calcium signalling pathway, cAMP signalling pathway, and PI3K-Akt signalling pathway as the most stringent pathways. After the 2nd month of irradiation, the G8 group shows a slight change in gene expression regulating antigen processing and presentation, cell adhesion molecules (CAMs) pathway, insulin signalling pathway, cGMP-PKG signalling pathway, calcium signalling pathway, cAMP signalling pathway, PI3K-Akt signalling pathway, mTOR signalling pathway, adipocytokine signalling pathway, toll-like receptor signalling pathway, and melanogenesis pathway. However, the G9 group has demonstrated glucuronic acid conversion, glutamatergic synapses, apoptotic signalling pathways, PI3K-Akt signalling pathway, Jak–STAT signalling pathway, ubiquitin-mediated proteolysis, ErbB signalling pathway, and ribosome as significant pathways than control (Figure 4A).

Similarly, the G10 group’s (8Gy) striatum was found to regulate the mTOR signalling pathway, adipocytokine signalling pathway, HIF-1 signalling pathway, insulin signalling pathway, Jak–STAT signalling pathway, and PI3K-Akt signalling pathway, compared to the control. Interestingly, the PI3K-Akt signalling pathway remains active in all irradiated groups (G7–G10: 3.4 Gy and 8 Gy of ⁵⁶Fe²⁶⁺), whereas the mTOR signalling pathway was observed in an active state for 2 months (G7, G8, and G10). The active phase of the PI3K-Akt signalling pathway and mTOR signalling pathway, in addition to other neuroinflammatory pathways, has depicted the activation of glial cells to repair neuronal damage induced by radiation. However, altered expression of glucose metabolic pathways has also reflected sequel damage or repair of the striatum over variable radiation exposure.

Furthermore, the active biological processes involved in regulating striatum function during different irradiation conditions were analysed through Gene Ontology. The top 10 upregulated biological processes are shown in Figure 4B. The differential gene expression of the G7 group shows the involvement of response to cAMP, anion transport, anion transmembrane transporter activity, apoptotic process, chemical homeostasis, substrate-specific transmembrane transporter activity, and MAPK cascade when compared with control. In the G8 group, other significant biological processes include movement of a cell or subcellular components, regulation of acute inflammatory response, apoptotic mitochondrial changes, MHC protein complex, and antigen processing. In contrast, the G9 group demonstrated regulation of T cell activation, interkinetic nuclear migration, positive regulation of apoptotic inflammatory response, glucocorticoid stimulus, etc. However, based on the dose effects, no significant difference in biological pathways was found between 3.4 Gy and 8 Gy after 2 months. In contrast, a substantial difference in biological pathways was observed between 3.4 Gy and 0 Gy, including the MHC protein complex, antigen processing, and MAPK cascade. Taken together, it can be inferred from the above analysis that as the irradiation dose range increases with time, the level of neuronal damage rises, leading to the activation of neuroinflammatory pathways and other linked pathways, postulating a peculiar cell signalling between neurons and immune cells.

To study the impact of heavy ion radiations on the cell signalling between neuronal and immune cells, we established brain in situ condition by culturing SH-SY5Y cells (neuron-like cells) and U87 cells (astrocytic glial cells) together, following a preliminary study based on primary neurons (see Supplementary File S1), as described previously by our team (Wang et al., 2014) and then irradiated with ¹²C⁶⁺ ions (1, 2, and 5 Gy). After irradiation, 24 h medium was collected and subjected to immune cells (THP-1: human leukaemia monocytic cell line, Jurkat: human leukemic T-cell line, and U937: human histiocytic lymphoma cell line) as prescribed markers of astrocyte activation. Our results showed that irradiated nerve cells produce differential effects on different immune cells. As shown in Figure 5A, U87 condition medium produced inhibitory effects on THP-1 proliferation at 2 Gy and 5 Gy radiation dosage, while SH condition medium produced no effect on THP-1 cells. However, the conditioned medium of U87 + SH co-culture irradiation has promoted the THP-1 cell viability with increasing radiation dose (2 Gy and 5 Gy). Thus, it speculates that irradiation causes glial cell damage and inhibits the THP-1 viability compared to the control group (0 Gy). In contrast, increased cell viability of THP-1 in the irradiated conditioned medium co-culture U87 + SH demonstrates that signal activation has occurred between neurons and glial cells to phagocytose neuronal debris.

Similarly, low U937 cell viability was observed in the U87 conditional medium after irradiation with 1, 2, and 5 Gy, while negligible radiation damage was observed on U937 cells in the SH medium. On the other hand, co-culture of U87 + SH conditional medium promoted U937 viability at 1 Gy dose; however, a significant decline in U937 cell proliferation was observed at 2 Gy irradiation, following a slight increase in the U937 cell viability with no significant difference at a dose of 5 Gy (Figure 5B), postulating that cytokines release after U937 damage in the nerve cells under co-culture conditions might be influenced by cytokine concentration. Additional investigation on Jurkat cells showed that irradiated U87 condition medium had inhibited Jurkat cell’s activation and proliferation at doses of 1, 2, and 5 Gy doses, whereas a co-cultured, post-radiated U87 + SH conditional medium promoted the activation and proliferation of Jurkat cells at a dose of 5Gy, as observed in our previous findings (Lei et al., 2015). Taken together, these findings indicate that dose-range is strongly linked to the differential expression of different immune cells. For instance, as shown in Figure 5C, THP-1 cell and Jurkat cell activation and proliferation were directly proportional to the increasing dose, whereas U937 cell viability was inversely proportional to the higher dose.

During an active inflammatory response, damaged astrocytes produce cytokines to recruit monocytes (THP-1) from the peripheral immune system and differentiate into macrophages (Chanput et al., 2014). A transwell migration assay was performed to evaluate the ability of these irradiated conditioned medium co-culture glial cells to recruit THP-1 cells and their differentiation into macrophages. Our results showed that a significantly low number of THP-1 cells (p < 0.001) migrated in the irradiated conditioned medium compared to the control (Figures 5D–F). Therefore, this indicates that neural cell injury due to irradiations had promoted the viability of the monocytes but reduced monocyte invasion and migration, as previously reported by our research group (Lei et al., 2015).

Additionally, to identify the impact of irradiated conditioned medium on immune cells, we also analysed the cytokines levels in the SH-SY5Y cells, U87 cells, and co-culture U87 + SH conditional medium. Interestingly, we observed a significantly higher level of IL-2, MIG, and MIP-1α (p < 0.05) in the SH-SY5Y cells, U87 cells, and co-culture U87 + SH conditional medium, indicating that cytokines release had promoted T-cell activation and IFN-γ secretion (Figures 6A,E,F). Similarly, significantly lower levels of IL-10, MIP-1β, and IL-12/IL-23 (Figures 6B–D) in the irradiated SH-SY5Y cells, U87 cells, and co-culture U87 + SH conditional medium (p < 0.05) hint at an aggravated immunological reaction in response irradiation. Therefore, based on our results, this complex interplay between neuronal and immune cells in response to heavy ion radiation reflects the dynamic and context-dependent nature of these interactions.