cells
Review
Control of Neuroinflammation through Radiation-Induced
Microglial Changes
Alexandra Boyd 1, Sarah Byrne 1, Ryan J. Middleton 1 , Richard B. Banati 1,2 and Guo-Jun Liu 1,2,*
1 Australian Nuclear Science and Technology Organisation, Sydney, NSW 2234, Australia;
boyda@ansto.gov.au (A.B.); sarahbyrne916@gmail.com (S.B.); rym@ansto.gov.au (R.J.M.);
rib@ansto.gov.au (R.B.B.)
2 Discipline of Medical Imaging & Radiation Sciences, Faculty of Medicine and Health, Brain and Mind Centre,
University of Sydney, Sydney, NSW 2050, Australia
* Correspondence: gdl@ansto.gov.au
Abstract: Microglia, the innate immune cells of the central nervous system, play a pivotal role in the
modulation of neuroinflammation. Neuroinflammation has been implicated in many diseases of the
CNS, including Alzheimer’s disease and Parkinson’s disease. It is well documented that microglial
activation, initiated by a variety of stressors, can trigger a potentially destructive neuroinflammatory
response via the release of pro-inflammatory molecules, and reactive oxygen and nitrogen species.
However, the potential anti-inflammatory and neuroprotective effects that microglia are also thought
to exhibit have been under-investigated. The application of ionising radiation at different doses
and dose schedules may reveal novel methods for the control of microglial response to stressors,
potentially highlighting avenues for treatment of neuroinflammation associated CNS disorders, such
as Alzheimer’s disease and Parkinson’s disease. There remains a need to characterise the response of





 microglia to radiation, particularly low dose ionising radiation.
Citation: Boyd, A.; Byrne, S.;
Middleton, R.J.; Banati, R.B.; Liu, G.-J. Keywords: neuroinflammation; microglia; TSPO; mitochondria; cytokines; antioxidants
Control of Neuroinflammation
through Radiation-Induced
Microglial Changes. Cells 2021, 10,
2381. https://doi.org/10.3390/ 1. Introduction
cells10092381 Ionising radiation (IR) as a diagnostic tool—such as X-ray, or positron emission
tomography (PET)—and therapeutic technique has been widely used for decades in the
Academic Editor: Fengru Tang pursuit of better health outcomes for patients [1]. Typically, these methods use lower
doses of ionising radiation, and are prescribed when the potential benefits to receiving
Received: 28 June 2021 the procedure outweigh the risks associated with IR [2]. However, fundamental to this
Accepted: 2 September 2021
practice is the acceptance of the linear-no-threshold (LNT) model; the understanding that
Published: 10 September 2021
ionising radiation initiates detrimental effects to human health in a manner proportional
to dosage [2,3]. This model, developed in the 1950s, arose from the extrapolation of the
Publisher’s Note: MDPI stays neutral
linear dose-response trend at higher doses and applying it to lower doses, where negative
with regard to jurisdictional claims in
published maps and institutional affil- effects have been presumed, but not observed [3,4]. The LNT model has been employed by
iations. regulatory bodies and accepted by both scientific and medical communities in the absence
of an alternate comprehensively proven model [5].
The acceptance of the LNT model may limit the potential of IR as a therapeutic tool
except in instances where the benefits heavily outweigh the perceived risk, for example,
radiotherapy. Although high doses of ionising radiation (HDIR) have been shown to
Copyright: © 2021 by the authors.
have adverse health effects, such as carcinogenesis [6,7], the presumption that low dose
Licensee MDPI, Basel, Switzerland.
ionising radiation (LDIR) would also have negative effects simply to a lesser degree is
This article is an open access article
unfounded [8,9]. In fact, hormetic effects have been demonstrated in numerous aspects
distributed under the terms and
conditions of the Creative Commons of human health; for example, sunlight is essential in vitamin D synthesis. However,
Attribution (CC BY) license (https:// high doses or prolonged exposures can result in sunburns and the development of skin
creativecommons.org/licenses/by/ cancers [10]. Additionally, data from both large-scale nuclear accidents [11,12], and the
4.0/). atomic bombings of Japan [13], do not fully support the LNT model, and there is a growing
Cells 2021, 10, 2381. https://doi.org/10.3390/cells10092381 https://www.mdpi.com/journal/cells
Cells 2021, 10, 2381 2 of 20
body of literature suggesting that exposure to LDIR may enhance putative neuroprotective
adaptive cellular pathways, such as increased antioxidant levels and reduced reactive
oxygen species, which may reduce inflammation within the CNS [14,15]. The increasing
availability and utility of IR, and the growing body of evidence suggesting the invalidity
of the LNT model, necessitates the reinvestigation of the current conceptions around the
safety and dose limitations of IR.
Microglia, the resident immune cells of the central nervous system (CNS), respond to
external stressors such as pathogenic invasion and injury by inducing inflammation [16–18].
During microglial activation, microglia are polarised from the M2 anti-inflammatory state
to the M1 pro-inflammatory state [16,19,20]. Alterations in the functional states of mi-
croglia in response to stressors are characterised by morphological changes and functional
plasticity [19,20]. The immune response that ensues is characterised by increased levels
of pro-inflammatory cytokines and reactive oxygen species (ROS) which promote the
degradation of damaged tissues and pathogenic invaders [21,22]. The M1 functional state
is associated with pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, whereas
the M2 functional state is associated with anti-inflammatory cytokines such as IL-4 and
IL-10 [23]. However, it has been shown that following microglial activation, both pro- and
anti-inflammatory genes are upregulated [18].
Notably, the expression of translocator protein (TSPO) is upregulated within the
mitochondria of activated microglia, and hence is often used as a biomarker of neuroinflam-
mation [24]. This inflammatory effect though to be beneficial to the body, as it is a protective
mechanism against disease. However, neuroinflammation has also been implicated in
many CNS diseases, such as Alzheimer’s disease [25–28], depression [29], and Parkinson’s
disease [30,31], indicating inappropriate chronic microglial activation. As microglia appear
to play a critical role in the onset and maintenance of neuroinflammation, the physiology
behind microglial activation and immune modulation pathways are of interest as potential
therapeutic targets [32]. This review will examine the contrasting characteristics of acti-
vated microglia when exposed to differing degrees of stressors, with a focus on ionising
radiation, to highlight the remaining uncertainties regarding microglial activation.
We acknowledge there is contention around both the term “neuroinflammation” and
the diseases it applies to [33–35]. A large portion of the scientific community has em-
braced the term, applying it to any condition where microglial and astrocytic activation
can be observed. This has led to the understanding that diseases such as Alzheimer’s
disease, Parkinson’s disease, and depression are “neuroinflammatory” diseases [29,35,36].
However, gene expression data indicate that these diseases are distinct from other known
inflammatory diseases [33–35]. Hence it best to restrictively use the term “neuroinflam-
mation” as a shorthand to describe the presence of microglia whose morphology or RNA
or protein expression profile is different from that ordinarily observed in health brain
tissue. Since under the term “neuroinflammation” microglial state changes (or “microglial
activation”) can be the consequence of a wide range of local or systemic immune system
responses, “neuroinflammation” should not be used as predictors of specific physiological
outcomes [33–35]. However, as the term neuroinflammation continues to be used by many
authors, this review, too, will refer all instances of microglial activation as to neuroinflam-
mation in its broader meaning but specify the context within which the term needs to
be interpreted.
2. Functional States of Microglia Altered by Stressors
Microglial cells play an important role in inflammation, brain development, and
the regulation of neuronal networks [37,38]. Historically referred to as the endogenous
macrophages of the CNS, this description is not completely comprehensive as it reflects only
one specific functionality of the cell and suggests that the mechanism of action for microglia
and macrophages are inherently the same. However, the initiation and maintenance of an
immune response is a major aspect of the function of microglial cells.
Cells 2021, 10, 2381 3 of 20
It is believed that microglia arise from primitive macrophages (myeloid progenitor
cells) in the embryonic yolk sac of mammals, before infiltrating the brain where they
differentiate and reside for life [39,40]. In the adult CNS, the microglial population does
not arise from further myeloid progenitor cells, instead the resident microglia self-renew
as needed and can rapidly proliferate in response to neural insults [41]. When a threat
is detected, such as a pathogen or radiation injury, microglia undergo morphological
transformations as they become “activated”. Traditionally, microglia were characterised
as either active (M1) or resting (M2); however, it is now understood that a spectrum of
microglial functional states exist [42,43]. Generally, active microglia adopt an amoeboid, less
ramified morphology, allowing them to become more mobile and phagocytotic, whereas
resting microglia have a smaller cell body and are highly ramified, allowing them to survey
the microenvironment [43–45]. Morphological changes such as cell area, perimeter and
ramification length are still frequently utilised in research as an indicator of the degree of
neuroinflammation [46,47].
A third morphology is gaining further scientific attention. Dystrophic microglia tend
to be small and de-ramified, with beaded or discontinuous processes [48,49]. The cause
of dystrophic microglia remains unclear; however, it has been hypothesised that they
are linked to ageing [16,48]. A recent study by Shahidehour et al. (2021) found that hy-
pertrophic (activated) microglial numbers, and not dystrophic microglia numbers, were
associated with ageing in the CA1 region of the hippocampus [49]. They found no differ-
ence in the percentage of hypertrophic microglia between neurodegenerative pathologies
(AD, Lewy body dementia and limbic-predominant age-related TDP-43 encephalopathy)
and age matched controls; however, in neurodegenerative pathology, 45% of microglia
were dystrophic, compared to 9% in the control [49]. Ethanol exposure has also been shown
to both reduce overall microglia numbers, whilst increasing the number dystrophic mi-
croglia in the hippocampus [50]. Interestingly, the researchers observed that the microglia
following ethanol exposure appeared “activated but not to an M1-like, amoeboid state”
highlighting the diversity of microglial functional states and how the M1/M2 classification
system may need revision [50].
In addition to morphological changes during microglial activation, there are nu-
merous alterations to bio-cellular pathways which promote an inflammatory immune
response. These pathways have primarily been established by exposing microglial cells
to lipopolysaccharide (LPS), an endotoxin derived from Escherichia coli. LPS, and other
stressors, which act on toll-like receptor 4 (TLR4) of microglial cells, initiating an inflam-
matory response [51,52]. This triggers microglial cells to become phagocytotic; engulfing
and degrading foreign materials [53]. It has recently been shown that inhibition of TLR4 in
an Alzheimer’s cell model promotes an M2 phenotype and improves neurological func-
tion [54]. The triggering receptor expressed on myeloid cells 2 (TREM2) has also been
implicated in the modulation of microglial phagocytosis, with some studies suggesting
that TREM2 function, and hence phagocytotic function, may be impaired in instances of
neurodegenerative diseases, particularly Alzheimer’s disease [55–57].
TLR4 signalling will lead to the downstream phosphorylation of Nuclear Factor
Kappa B (NF-κB) inhibitory protein, promoting the expression of pro-inflammatory genes
and therefore the expression of pro-inflammatory proteins [58–62]. Among the more
commonly known ones are cytokines IL-1β [63], IL-6 [64], and TFNα [65] which play a
role in triggering cell cycle arrest and apoptosis [66], the initiation of neurotoxicity [67],
and inflammatory signalling [62,68]. There is also emerging evidence that some pro-
inflammatory cytokines may enhance the dopaminergic differentiation of neural stem
cells, i.e., they may possess neurogenic properties [69]. The transcription of enzymatic
genes, such as iNOS, and apoptotic genes, such as Fas-ligand, results in the translation
of proteins which play an active role in inflammation and cellular death [70–72]. In
mice models of neurodegeneration, it has been observed that the downregulation of
homeostatic microglial genes correlates with neuronal loss, whereas the upregulation of
disease associated microglial genes did not [73]. The one exception to this was the APOE
Cells 2021, 10, 2381 4 of 20
gene, which directly correlated with neuronal loss [73]. In fact, APOE4 is a known genetic
risk factor for Alzheimer’s disease [74]. The study also found that early AD pathology
in the human brain was only associated with a loss of homeostatic genes, but not the
gain of any disease related genes. This suggests interspecies variability in microglial gene
expression during pathological states, meaning the results of murine studies may not
translate into the clinic [73].
Increased reactive oxygen and nitrogen species (RONS) concentration are also a
hallmark of neuroinflammation [75]. In microglia, the majority of RONS are reactive
oxygen species generated via NADPH oxidase; however, they can also originate from other
intra- and extracellular sources [76,77]. Interestingly, some studies show that LPS- and
a-Synuclein-induced neuroinflammatory responses are attenuated in NOX2 (an isoform
of NADPH oxidase) knockout mice, indicating that NOX2 may play a role in microglial
activation [78]. NADPH oxidase has also been implicated in cognitive dysfunction in
experimental autoimmune encephalomyelitis, being demonstrated to prevent long term
potentiation [79]. The iNOS enzyme in microglia and macrophages is responsible for the
production of nitric oxide (NO), a precursor to reactive nitrogen species (RNS) which,
in conjunction with reactive oxygen species, result in oxidative damage to lipids and
proteins [80]. NO can also prevent cell division, often leading to cell death, by inhibiting
an enzyme required for DNA synthesis, or by directly causing double stranded DNA
breaks [81–84].
On the other hand, microglia display an array of neuroprotective effects. Immune
surveillance is constantly occurring under homeostatic conditions, during which microglial
processes and filopodia randomly survey the external environment searching for cues
which may trigger an immune response [85,86]. Microglia also have a role in synaptic
pruning and promoting neurogenesis [87–91]. Synaptic pruning allows for the removal
of weaker synaptic connections, which promotes the development of stronger pathways,
allowing for clear and direct signal transduction [92]. The importance of microglia in synap-
tic pruning is demonstrated through the knockout of Cx3cr1 receptors, a critical receptor
for microglial migration. Cx3cr1 knockout in mice results in immature brain circuitry,
which possess the electrophysiological hallmarks of undeveloped synaptic function [89].
A transcriptomic analysis also demonstrated that the microglial phagocytosis of human
apoptotic cells initiated the expression of neurogenic-related genes, strongly suggesting
that microglia modulate the process [93]. Recently, one study found that the supernatant of
M2 microglial cells (containing molecule 15-deoxy-∆12,14-prostaglandin J2) promoted neu-
rogenesis and oligodendrogenesis [94], whereas another demonstrated the direct contact
between a microglial cell and neuronal dendrites promoted synaptic formation [95]. It has
also been shown that microglia are necessary to learning-dependent synaptic formation,
and this plasticity is regulated via brain derived neurotrophic factor [96]. It is therefore
clear that microglial have a role in neurogenesis.
3. Impact of Ionising Radiation on Healthy Brains by Altering Microglial
Function States
Phenotypic changes to microglia can be achieved by the application of ionising radi-
ation, the effects of which are widely believed to be dependent on the dosage and dose
schedule. Knowledge of the effects of ionising radiation, primarily established through
large scale nuclear events, indicate that HDIR is detrimental to human health [97,98]. The
linear no threshold model arose from extrapolating this knowledge and applying to it
LDIR [99]. The LNT model is still widely used by many radiation protection organisations
today; however, there is an increasing need to re-evaluate this on the basis of recent evi-
dence highlighting the potentially positive effects of LDIR. Currently, there is no consensus
on what dose constitutes LDIR or (HDIR). Often, a low dose is defined as below 100 mSv;
however, this does not take into account dose rate, cumulative dose or potential interspecies
variability. As such, a variety of doses and their effects have been included.
Aside from the widely accepted risk of carcinogenesis, HDIR has detrimental effects
on the human CNS, having been linked to the onset of cognitive dysfunction [100–103],
Cells 2021, 10, 2381 5 of 20
deficits of spatial-temporal learning, and reduced memory (2–54 Gy) [104–108]. Ionising
radiation has also been shown to cause demyelination (see review [109]), and to disrupt
neurogenesis (see reviews [110,111]). Additionally, a reduction in functional connectivity
in the anterior cingulate cortex and right insular region has been observed following
radiation therapy in nasopharyngeal carcinoma patients, where 68–70 Gy was administered
over 30–33 fractions [112]. These deficits tend to peak around 4 months post radiation
treatment [113], and can be irreversible. However, it is important to note that most human
epidemiological studies arise from opportunity and, therefore, in most human studies the
disease which is being treated may be a confounding factor. Similarly, a lot of long-term
effects are unknown in these cases due to the typically shorter life span of the patients.
Cell and animal research continue to help bridge the gap in areas which human
studies cannot explore. Various studies have shown a range of microglial responses to
high dose ionising radiation; notably that irradiation using a high dosage will elicit a
neuroinflammatory response [18,114–118]. A dose of 0.5 Gy has been shown to increase
the number of microglia in the hippocampus, compared to control and low dose ionising
radiation (0.063 Gy); however, the microglia were less ramified than before [118]. A similar
increase in microglial density has been found in the cerebellum following a 6 Gy dose [119].
Osman et al. recently observed that a dose of 8 Gy on the juvenile murine brain induced
transient microglial activation, as demonstrated through changes in microglial morphology
and density [18]. Microglial activation was also associated with a transient increase in
apoptotic cell levels, as well as a simultaneous increase in both pro- and anti-inflammatory
genes. Notably, the effects of the ionising radiation tended to peak around 6 hrs, after
which they began to decline [18].
Often linked with an increased microglial density is an increase in reactive oxygen
and nitrogen species production, which leads to protein oxidation and lipid peroxida-
tion [117,120–124]. Oxidative stress results in an increase in DNA damage such as double
stranded breaks and concomitant decrease in DNA repair proteins [125] and diminished
antioxidant enzyme activity [126]. However, one study contrasts this, showing that a dose
of 200 mGy may reduce lipid peroxidation within the brain, with associated increases
in catalase and antioxidant concentrations [127]. Pro-inflammatory cytokines such as
IL-1β, TNF-α, and IL-6 have all been shown to be increased after irradiation and play
a role in the inhibition of neurogenesis and further promotion of inflammation (refer
to Figure 1) [106,117,119,128–131]. Other observed reactions that are of interest include
changes in mitochondrial membrane potential and permeability [132], increased blood–
brain barrier permeability [119,133], and the induction of pyroptosis [134,135]. Crucially, it
has been shown that the transcriptome of microglia that have been exposed to high dose
radiation is significantly similar to the M1 classical activation phenotype [136], indicating
that HDIR activates microglia. Further evidencing the role of HDIR activated microglial
in neuroinflammation are recent studies which demonstrate that acute pharmacological
microglial depletion following radiation exposure alter the neuroinflammatory response
and can prevent cognitive deficits [102,137].
It has previously been shown that ionising radiation alters the brain architecture
in mice. In the hippocampus, ionising radiation has resulted in reductions in dendritic
complexity and number, and has altered the concentration of synaptic proteins [138].
Recently, microglia have been associated with radiation-induced synaptic loss. Male
mice exposed to 10 Gy IRexhibited a significant decrease in immature spinal density;
however, the knockout of complement receptor 3 (CR3) was neuroprotective and prevented
this loss [139]. Interestingly, female mice (knockout and wildtype) did not experience
radiation induced decreases in spinal density, and also displayed a significantly higher
number of intersections basally when compared to male mice, suggesting a sex-dependent
effect [139]. This study also observed changes in microglial activation markers following
the IR; however, not morphological changes, suggesting that morphological changes may
not be a reliable indicator of microglial activation [139]. HDIR may also induce neuronal
apoptosis by causing cell cycle arrest at the G2 and M checkpoints [117]. This study
Cells 2021, 10, x FOR PEER REVIEW 5 of 19 
 
confounding factor. Similarly, a lot of long-term effects are unknown in these cases due to the 
typically shorter life span of the patients. 
Cell and animal research continue to help bridge the gap in areas which human studies 
cannot explore. Various studies have shown a range of microglial responses to high dose ion-
ising radiation; notably that irradiation using a high dosage will elicit a neuroinflammatory 
response [18,109-113]. A dose of 0.5 Gy has been shown to increase the number of microglia 
in the hippocampus, compared to control and low dose ionising radiation (0.063 Gy); how-
ever, the microglia were less ramified than before [113]. A similar increase in microglial den-
sity has been found in the cerebellum following a 6 Gy dose [114]. Osman et al. recently ob-
served that a dose of 8 Gy on the juvenile murine brain induced transient microglial activation, 
as demonstrated through changes in microglial morphology and density [18]. Microglial acti-
vation was also associated with a transient increase in apoptotic cell levels, as well as a simul-
taneous increase in both pro- and anti-inflammatory genes. Notably, the effects of the ionising 
radiation tended to peak around 6hrs, after which they began to decline [18]. 
Often linked with an increased microglial density is an increase in reactive oxygen and 
nitrogen species production, which leads to protein oxidation and lipid peroxidation [112,115-
119]. Oxidative stress results in an increase in DNA damage such as double stranded breaks 
and concomitant decrease in DNA repair proteins [120] and diminished antioxidant enzyme 
activity [121]. However, one study contrasts this, showing that a dose of 200 mGy may reduce 
lipid peroxidation within the brain, with associated increases in catalase and antioxidant con-
centrations [122]. Pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 have all been 
shown to be increased after irradiation and play a role in the inhibition of neurogenesis and 
further promotion of inflammation (refer to Figure 1) [101,112,114,123-126]. Other observed 
reactions that are of interest include changes in mitochondrial membrane potential and per-
meability [127], increased blood–brain barrier permeability [114,128], and the induction of py-
roptosis [129,130]. Crucially, it has been shown that the transcriptome of microglia that have 
Cells 2021, 10, 2381 been exposed to high dose radiation is significantly similar to the M1 classical activation p6 hofe2-0
notype [131], indicating that HDIR activates microglia. Further evidencing the role of HDIR 
activated microglial in neuroinflammation are recent studies which demonstrate that acute 
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tocrhye mresopkoinnesem aRndN cAane xpprerevsesniot cnoogcnciutirvrei ndgef1ic-witse [e9k7,p1o32st].e xposure [117].
 
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number of activated microglia, which increases oxidants and pro-inflammatory cytokines, creating 
at nheeunruominbflearmofmaacttiovrayt esdtamtei. croglia, which increases oxidants and pro-inflammatory cytokines, creating
a neuroinflammatory state.
Additionally, rodent models have allowed the investigation of the CNS effects of
 prenatal radiation exposure. The paucity of human studies have suggested there is poten-
tially an increased risk of cognitive and health effects on a foetus; however, the Centers for
Disease Control and Prevention (CDC, USA) has acknowledged that an acute radiation
dose of <100 mGy has no observable non-cancer effects [140–143]. The majority of human
studies which involve HDIR come from A-bomb survivors, and these indicate the potential
for prenatal IR to cause mental retardation and microcephaly [144]. Animal studies have
highlighted dose and pregnancy stage dependent effects of HDIR causing DNA damage,
alterations to cell cycle checkpoints and apoptosis in the neocortex [145–147], and have
identified behavioural/cognitive changes [145].
The effects of LDIR have been observed to be quite different and conflicting. There
is a growing body of evidence which suggests that LDIR may be anti-neuroinflammatory
(refer to Figure 1). This hypothesis is termed radiation hormesis. Human cells grown
under reduced background radiation manage the stress of acute irradiation at high dose
less efficiently than cells cultured under normal background radiation [148], supporting
the theory that mild radiation exposure stimulates an adaptive response. Irradiated fruit
flies and rodents also have shown enhanced immune systems and extended lifespans
compared to non-irradiated controls [149–152]. One murine lifetime study found that an
exposure of single 0.063 Gy radiation significantly reduces the risk of the development
of many types of tumours, including pheochromocytomas, adenomas, insulinomas, and
adenocarcinomas, compared to non-exposed controls [152]. LDIR is also thought to confer
protection to cell functioning, molecular structures, synapses and key brain mechanisms
such as neurogenesis, as well as inducing reparative functions (see review [153]). These
theories have been supported by observed physiological responses LDIR. The suppression
of ROS is one such example [154,155]. Studies of occupationally exposed workers found
that chronic LDIR (0.1–8.4 mGy per month) was associated with an increased resistance to
oxidative stress [156,157]. This effect has also been observed at higher acute doses, with
acute 0.2 Gy IR increasing antioxidants in the blood and tissues of rats [127]. Increases
in anti-inflammatory cytokines such as IL-4, IL-10, and reductions in inflammatory cy-
Cells 2021, 10, 2381 7 of 20
tokines such as TFN-α have been observed at 50 mGy [158]; however, this effect has also
been observed at higher doses of up to 1 Gy [25,159,160]. Of note, one study found that
100 mGy actually increased inflammatory cytokines [161], and another found that 100
mGy may have a negative effect on cognition, although noted further investigation is
required [108]. Notably, 100 mGy appears to disrupt the BBB, which is a key aspect of
neuroinflammation [74,162].
A recent study by Ung et al. (2020) specifically examined the effects of a singular
radiation event on both murine glial cells and murine behaviour [118]. At the “high” dose
of 0.5 Gy, there was an observable decrease in acoustic startle response, exploration, and
rearing at 12 months post exposure. At 24 months post exposure, there was a significant
increase in the number of microglial cells in the dentate gyrus, and a reduction in both
astrocyte number and complex morphology. However, mice exposed to a singular dose
of 0.063 Gy radiation had an increased acoustic startle response, increased exploratory
behaviour, and increased rearing at 18 months post exposure, and had significant increases
in microglial ramification at 24 months (compared to both the control and other irradiated
groups of 0.125 and 0.5 Gy) despite no observable increase in microglial density. The
astrocyte morphology of this group was also not significantly different to the control group.
This study clearly demonstrates the potential for LDIR to be anti-neuroinflammatory in
comparison to HDIR [118]. A complementary study by Hladik et al. (2020) observed the
effects of a single radiation event on cAMP response element-binding protein (CREB) sig-
nalling; a transcription factor involved in memory formation, neuroplasticity and amyloid
processing [163]. The CREB pathway, and other associated pathways, were found to be
“activated” (as determined by alterations in hippocampal protein levels) by a dose of either
0.063 Gy or 0.125 Gy, whereas these pathways were “deactivated” by a dose of 0.5 Gy.
Conditioning, learning, and long-term potentiation were found to be activated by 0.063 Gy
or 0.125 Gy and were deactivated at 0.5 Gy. Additionally, a dose of 0.125 Gy was found
to deactivate apoptosis. This data further supports the concept that LDIR may allow for
neuroprotective cellular adaptive responses in comparison to the detrimental effects of
higher doses [163], a premise which is supported by our recent study in press at Frontiers
in Cell and Developmental Biology, which demonstrated that LDIR (10 mGy) may enhance
neuroprotective pathways in the healthy brain [164].
However, radiation hormesis tends to be poorly supported by most human studies.
An 11 million person cohort study found that the LDIR dose from a computed tomography
(CT) scan (~40 mGy) during childhood and adolescent correlates with an increased inci-
dence of cancers [165]. A recent meta-analysis examining a potential link between low dose
ionising radiation exposure in adulthood and cancer similarly found that, after excluding
studies with potential biases from the null, there was still a positive risk estimate reported
by many studies [166]. Another systematic review found no positive effects of LDIR on
neurodevelopment and cognition; however, it indicated that the evidence of adverse effects
is “limited to inadequate” [167]. In a population of adults with congenital heart disease,
a greater exposure to LDIR from cardiac procedures correlates with an increased cancer
incidence [168]; however, the population of “adults without cancer” were significantly
younger therefore had less comorbidities than the “adults with cancer population.” A re-
view by Lumniczky, Szatmári, and Sáfrány (2017) found that LDIR could result in cognitive
defects and other unfavourable outcomes in both human and animal populations, and
resulted in the induction of different molecular and cellular mechanisms [169]. The authors
called into question the safety of LDIR for diagnostic purposes [169]. Another review by
Tang and Loganovsky (2018) concluded that LDIR (<100 mGy) or low dose rate ionising
radiation (<6 mSv/hr) may or may not induce cancer, depending on a variety of factors
including demographics, lifestyle, and diagnostic accuracy [170]. However, LDIR may in-
crease incidences of vascular diseases, cognitive and mental health disorders, eye diseases,
and other pathologies, whilst reducing cancer mortality and mutations, and increasing
longevity [170]. Despite more studies beginning to investigate radiosensitivity [171], it
remains unclear what effect LDIR truly has on different organs or tissues. One important
Cells 2021, 10, 2381 8 of 20
effect which cannot be ignored is the potential effect of LDIR on cellular senescence. Car-
bon ion irradiation (1 mGy) can lead to premature senescence in human lung fibroblasts;
however, this effect was not observed following 1 mGy gamma irradiation [172].
Cells 2021, 10, x FOR PEER REVIEW Given that ionising radiation can induce the polarisation of resting microglia8 ionft o19a n
 activated state, there is the potential to explore different doses of ionising radiation as a
tool to trigger the switching between activation states; in particular, from inflammatory
to anti-inflammatory state by LDIR. We know that the functional state of microglia is dy-
infnlaammmic,aatonrdyt, hMat1c hpahnegniontgypthee tehnevni raoltnemr ethnet  ceannvbireoanmmeencht atnoi strmigogfemr aa nsiwpuitlcahti nbgactkh etmo t[h1e7 3].
M2It phhaesnaoltryepaed y[1b6e7e].n Tdheemreo ins satlrsaot eedvitdheantcteh teor esuisgpgeostet nthtiaatl tthoec monicvreorgtlmiali carcotgivliaatiionnt ostaantei n-
canfl abme maatnoirpyu, lMat1edp hbeyn roetpyepaetethde cnhaalltleerngthees ewnivthir osntimeunlit tthoattr iigngfleureancsew fiutcthurbea mckictorotghleiaMl 2
behpahveinooutry puep[o1n7 4s]u. Tbsheeqrueeisnta lsstoreesvsi d[e1n6c8e]. toThsuergegfeosrte,t hfauttuthree mstiucdroiegsl iaslhaocutilvda tiniovnessttiagtaetcea n
whbeethmera nchipaullleantegdinbgy mreicpreoagtleiad wchitahl ldenifgfeersenwti tdhossetism aunldi  tdhoastei nscflhueednucleesf uotfu iroenmisiincgro rgaldiaial -be-
tiohna cvaino uernuabploen thsue bcsoenqturoenl ot fs ttrhees sm[1ic7r5o]g. lTiahle fruenfocrteio, nfuatlu srteatsetu, adsi eist issh ao uplodteinvtieasl ttihgearteapwehuettich er
tooclh faolrle neguinroginmfliacmromglaiatiowni-tahsdsoifcfieartendt pdaotsheosloagnidesd souscehs cahse Adluzlheesiomfeiro’ns iasnindg Prardkiiantison ’csa n
diseenaasbel. e the control of the microglial functional state, as it is a potential therapeutic tool for
neuroinflammation-associated pathologies such as Alzheimer’s and Parkinson’s disease.
4. Impact of Low Dose Ionising Radiation on Neurodegenerative Diseases 
4. TImhep pacotsoitfivLeo ewffeDctoss oef ILoDniIsRi nogn Rmaoddiealtsi oonf AonlzNheeiumreord’se dgeisneearsaet ihvaeveD biseeeans seesen in mice, 
particulTahrley pino stirtaivnesgeeffneicct sAoDf  LfeDmIRaloe nmmicoed (erlesfeorf tAo lFzhigeuimree 2r)’s [1d6is9e]a. sAe dhoavsee obfe e1n00s emenGyin im-ice,
propvaerdti cluoclaormlyotionrt aractnivsgiteyn iinc AlDzhfeeimaelre’sm-liikcee t(rraenfsegretnoicF i(gTugr) efe2m) a[1le7 6m].icAe, dimosperoovfe1d0 0thmeirG y
griipm sptrreonvgetdh laoncodm reodtourceadc tAivβitxy-4in0 lAevlzehlse.i mTheer ’ssa-mlikee dtroasne signe mniacle(T Tgg) fmemicea lheamd ifceew, i mefpfercotvs,e d
butth deiidr gsirginpifsitcraenntglyth readnudcere mduoctoerd cAooβrxd-i4n0atlieovne.l sIn. tTehreestsianmgley,d tohsee hiinghmear ldeoTsge omf i5c0e0h madGfye w
alseof fiemcptsrobvuetdd liodcsoimgnoitfiocra ancttliyvirteyd iun cTegm feomtoarlecso oforrd ionpaetnio mn.aIznet etersets atinndg lfyo,rt hTeg hmigahleesr idn otshee of
Y-m50a0zem, iGmyparlosvoeidm mporotovre cdoloorcdoinmaotitoonr ianc tTivg ifteyminalTesg afnedm raeldesucfoedr  oApβexn-4m0 alezveetles.s At arngudafbolry,T g
them maolesst iimn pthoertaYn-mt taazkee,awimapyr forovmed thmiso sttourdcyo iosr tdhianta, twioitnhoinutT rgadfeiamtiaolne,s Tagn fdemreadleu mceidceA hβavxe-4 0
higlehveer lAs.βA lregvuelasb tlhy,atnh Tegm mosatleim mpicoer;t ahnotwtaekveear,w raadyifartoiomn trheidsuscteudd ythiessteh laetv,ewlsi tihno tuhter faedmiaatlieo n,
TgT mgifceem, walheicmh iccoerrhealavteedh iwghitehr dAeβcreleavseedls mthicarnogTlgialm aaclteivmatiicoen; ahso dweetevremr,inraeddi abtyi oCnDr6e8d urec-ed
cepthtoers eslteavineilnsgin [t1h6e9]f.e mReacleenTtglym, ircaet, wmhoidcehlsc oorfr eAlaDte dwweriteh adlesocr esahsoewdnm itcor ohgalviael aimctipvraotvioend as
medmetoerrym pinerefdorbmyaCnDce6 8inr erceesppotonrsest taoin ai nhgig[h17e6r ]d. oRseec eonf tiloyn, risaitnmg oraddeilastoiofnA oDf 2w Gerye/dalasyo fsohro 5w n
datyos,h wavitehiomupt rionvceredamsinegm onreyurpoeirnffolarmmamnacteioinn roers paomnysleotiod alohaidg h[e1r70d]o. sLeDoIfRio hnaissi naglsroa dbieaetnio n
shoowf 2nG toy /pdroamy footre 5and aMys2, mwoitrhpohuotloingcyr eians LinPgSn tereuartoeidn flmaimcem matiicornogolriaalm (ByVlo2i)d cleollasd [1[17717].] . LDIR
has also been shown to promote an M2 morphology in LPS treated mice microglial (BV2)
cells [25].
 
Figure 2. Low dose ionising radiation strengthens immunity in the healthy brain and reduces neu-
Figure 2. Low dose ionising radiation strengthens immunity in the healthy brain and reduces neu-
rodroegdeengeernaetriavteiv deidseisaesaes ebyb ychchanangginingg mmicicrroogglliiaall ffuunnccttiioonnaall ssttaatteess.. AAh heeaaltlhthyyb rbarianine xepxopsoesdetdo tloo wlodwo se
dosioen iiosninisginrga driaadtiioatniomna myaeyx peexrpieerniceencaen tain-itni-flinamflammamtoartyoreyff eecfftesc, tws,h werheearseians aind ae gdeengeernaetrivaetivbera binra, isnu, ch
sucahs ains Ainl zAhlezihmeeimr’serd’sis deiasseea,sleo,w lodwo sdeoisoen iiosinnigsinragd riaadtiioantimona ymlaeys sleensstehne tsheev eserivtyeroitfyn oefu nroeiunrfloainmflmamat-ion
maatniodnp arnodm portoema oshtei fat tsohwifta rtdows armdso rae m“noorerm “nalo”rmbrali”n bernavinir oenvmireonnt.ment. 
HuHmuamn acnascea ssteusdtiueds iheasvhea uvseeuds leodwl odwosde oioseniisoinngis irnagdiraatdioina taios na atrseaattmreeantmt feonr tnfeourrno-eu-
dergoedneegraetniverea dtiivsoerddiesrosr d(reerfser( rteof eFrigtourFei g2)u. rTeh2e) .mTohset mweolslt-kwneolwl-knn iso wa nseirsieas soefr aiersticolfeas rftoicl-les
lowing a patient with end stage AD. After 2 CT scans (~40 mGy each) to detect anatomical 
changes, the patient displayed several behavioural changes noticed by her family and car-
ers including signs of old memory return, improved motor function, and short three- to 
five-word sentence formation [172]. The patient then began to receive a CT scan approxi-
mately every 2 weeks. Interestingly, following the fifth CT scan the patient exhibited sig-
nificant decrease in cognitive and motor abilities, potentially demonstrating the fine 
 
Cells 2021, 10, 2381 9 of 20
following a patient with end stage AD. After 2 CT scans (~40 mGy each) to detect anatom-
ical changes, the patient displayed several behavioural changes noticed by her family
and carers including signs of old memory return, improved motor function, and short
three- to five-word sentence formation [178]. The patient then began to receive a CT scan
approximately every 2 weeks. Interestingly, following the fifth CT scan the patient exhib-
ited significant decrease in cognitive and motor abilities, potentially demonstrating the
fine balance between both dosage and dose rate on whether radiation has advantageous
or deleterious effects. The patient recovered from this setback, and continued to receive
frequent, though more spaced out, CT scans [178–180]. Additionally, having observed these
positive effects on his wife, the partner of the Alzheimer’s patient opted to receive CT scans
to treat his Parkinson’s disease [179]. He received a CT scan every few months. The patient
was able to reduce the dose of his medication, his tremors reduced, he was less constipated
and his vision improved [180]. These promising results prompted a small pilot study
where CT scans were used on four patients with AD [181]. Minor quantitative changes
were observed; however, there were “remarkable improvements” in qualitative measures
such as communication and behavioural changes. One of the four patients showed no
improvement [181]. Together, this case and pilot study show the positive effects low dose
radiation may confer on Alzheimer’s disease; however, until we can better control and
understand LDIR as a treatment, it is not feasible to use therapeutically.
It is also important to consider the effects of radiation in multiple sclerosis (MS), where
the immune system is already under the stress of an autoimmune condition. There is some
evidence which may suggest that ionising exposure may represent a greater risk in MS
than in healthy individuals. A clinical trial on total lymphoid irradiation (TLI) (19.8 Gy) for
the treatment of MS found that irradiation reducing lymphocyte numbers to <900 mm−3
slowed the progression of the disease [182]. A follow-up found that any toxicity was
“mild and transient” and suggested that the benefits of the treatment outweighed the
disadvantages; however, it did indicate that menopause was induced in two patients and a
staphylococcal pneumonia infection in another [183]. A further statement from the authors
discussed the causes of five deaths among the cohort. All of the deceased were in a high
lymphocyte, poor prognosis category, so it is plausible that the deaths may have occurred
regardless; however, there is also the possibility that the risk for serious infections is
increased after TLI [184]. The patient with the induced staphylococcal pneumonia infection
died from aspiration, providing merit to the latter theory [184].
Additionally, a 2013 case report suggested that conventional doses of ionising radiation
used to treat meningioma induced the onset of multiple sclerosis in a 43-year-old woman,
suggesting a potential connection but not establishing a causal relationship [185]. Other
case and cohort studies have indicated a similar trend [186–189]. One cohort study even
found that X-ray exposure site was relevant, with chest X-rays, skull X-rays, and brain CT
scans all aligning with a higher incidence of MS [190]. Conversely, there are studies that
indicate that there is an inverse relationship between IR, specifically ultra-violet B (UVB)
exposure, and the development of MS [191,192]. Gamma Knife Radiosurgery (85 Gy) has
been used to treat trigeminal neuralgia in MS patients, with 82% of patients reporting
a reporting a reduction in pain following one treatment [193]. However, the authors
acknowledge this needs to be explored further, with increased radiation toxicity being
observed by some studies [194]. One murine study observed that repeated 0.5 Gy doses
(to a dose of 10 Gy) of gamma radiation reduced many autoimmune symptoms, including
splenomegaly, lymphadenopathy, and proteinuria [195]. It therefore remains unclear the
effects of ionising radiation in pre-existing immune conditions.
5. TSPO as a Biomarker for Changes in Microglia
To quantify microglial activation, appropriate biological markers are necessary. These
biomarkers can take on a variety of forms, such as cell receptors or cytokines. The most
commonly used marker of microglial activation is ionised calcium binding molecule 1
(Iba1), also known as allograft inflammatory factor 1. In the CNS, it is expressed solely by
Cells 2021, 10, 2381 10 of 20
activated microglial cells and is responsible for membrane ruffling and phagocytosis [196].
Iba1 is highly conserved across species and can be easily detected through anti-Iba1 anti-
bodies [197].
Pro-inflammatory and anti-inflammatory cytokines and chemokines can also be ex-
amined to elucidate the current activation state of microglia. Increased concentrations of
pro-inflammatory molecules such as IL-6, IL-1β, IL-18, TNF-α, IFN-γ, CCL5, or GM-CSF
all indicate a greater proportion of activated microglia, and hence likely a greater degree of
neuroinflammation [198]. Opposingly, higher concentrations of IL-4, IL-10, TGFβ, or CCL22
all signify an anti-inflammatory environment [198]. However, cytokine and chemokine
analyses are often used to complement Iba-1 data, rather than as standalone data, as they
are not “neuroinflammation” specific and often require the use of homogenised tissues,
leading to spatial information being lost [199]. Therefore, these molecules tend not to be
the best biomarker of neuroinflammation.
Mitochondrial translocator protein 18 kDa (TSPO), like Iba-1, has been shown to be
upregulated in microglia under conditions of stress and pathology [200–203] and serves as
a biomarker of neuroinflammation [204,205], particularly for in vivo molecular imaging
such as a PET scan [206]. TSPO has a low basal expression in the central nervous system,
predominantly by endothelial cells [207]. Although its exact function remains unclear,
TSPO knockout mice have both helped to disprove its role in cholesterol translocation and
steroidogenesis [208–211], and highlight a role in microglial activation and mitochondrial
function [212]. TSPO is an attractive target for studying the effects of ionising radiation as
it has been implicated in ROS production and ROS-mediated oxidative damage [213,214],
and the addition of TSPO ligands, such as PK11195 or Midazolam, have been shown
to reduce pro-inflammatory gene expression, accompanied by a reduction in activated
microglia [215–217]. Manipulating TSPO expression or function will allow the understand-
ing of the link between mitochondrial function and neuroinflammation to strengthen,
supporting the future development of therapeutics targeting these elements. The use of a
TSPO knockout model to observe the behaviour of TSPO under varying conditions, and
the potential impact on the CNS microenvironment, provides a unique tool to characterise
microglial response in the presence and absence of TSPO to varying stressors and would
be invaluable to future study [209]. Our recent study demonstrates that there are decreases
in TSPO and Iba1 mRNA and protein levels in brain, and proinflammatory cytokine IL6 in
blood plasma, following 10 mGy IR, and increases in TSPO protein expression at 2 Gy in
the brains of healthy mice and in primary cultured microglia [164]. As the levels of neu-
roinflammation in the healthy brain are minimal, there is little inflammation to be reduced
by low dose radiation. However, the clear trend towards downregulation of TSPO and
Iba1 expression indicates that LDIR may reduce microglial activation, and hence neuroin-
flammation. Further investigations should be undertaken in models of neurodegenerative
diseases, where elevated levels of neuroinflammation are observed.
One potential downside, elucidated by a recent study, is that neuronal activity in-
creases TSPO levels within the brain, specifically in neurons, meaning it may be an unreli-
able measure of glial activation [218]. Additionally, the post-mortem brains of late stage AD
and Dementia with Lewy Bodies have shown similar TSPO levels to age matched controls,
and even showed a reduction in some areas such as the substantia nigra [219]. Here, a
reduction in TSPO may not indicate a reduction in neuroinflammation, rather may reflect
“dystrophy, senescence and death, or dysfunction of mitochondria in the microglia” [219].
Another recent study concluded that whilst TSPO effectively marks activated microglia,
it is not a predictor of neuronal loss, and as such only marks neuroinflammation and not
neurodegeneration [220]. Finally, there is evidence to suggest that TSPO ligands may bind
to plasma proteins, and therefore are unavailable to bind to TSPO, affecting the accuracy of
PET scan results regarding neuroinflammation [221].
Cells 2021, 10, 2381 11 of 20
6. Conclusions and Future Directions
As the innate immune cells of the central nervous system, microglia facilitate the
initiation and maintenance of basic immunity and neuroinflammation. The activation of
microglia through stressors such as radiation prompts the transcription of pro-inflammatory
genes, leading to the release of molecules such as IL-6 and TFN-α and the adoption of an
ameboid, phagocytotic morphology. However, there is a growing body of evidence that
LDIR may not act as a stressor, rather that LDIR may confer neuroprotection. Whilst the
molecular mechanisms behind this are largely unknown, many cellular and animal studies
have found LDIR promotes longevity, neurogenesis, and cognition, while decreasing ROS
production. There is paucity of human studies on the effects of LDIR on microglia, and
hence neuroinflammation. As such, the current understanding of radiation hormesis is poor.
As highlighted throughout the review, the dosage which divides the positive and negative
effects of radiation is hard to determine, with many studies providing conflicting results.
Furthermore, the duration (transient or indefinite) of any potential positive outcomes is
undetermined, and the interactions between different doses and exposure frequencies
are under-explored. It may be that case that repeated “low dose” treatments will accrue
and transform a positive effect into a detrimental one, as seen in the case study where CT
scans were used to treat a patient with AD [178]. Whilst the case studies of a woman with
AD and a man with PD show promise in the potential treatment of neuroinflammatory
conditions [179], the large cohort study of people who received a CT scan in childhood
or adolescence indicated an increased cancer risk [165]. It remains unclear whether the
effects of LDIR are beneficial to or adversely impact human health. Further animal and cell
work is required to elucidate the mechanisms behind the observed microglial mediated
neuroprotection, and research needs to be undertaken regarding dose-rate, age when
exposed (adulthood vs adolescence) and whether the impact is not only beneficial in
disease states but also to a healthy population.
Author Contributions: Conceptualization, G.-J.L.; writing—original draft preparation, A.B. and S.B.;
writing—review and editing, G.-J.L., R.J.M., and R.B.B. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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