Sutherland, B.M. et al. (2000), "Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation.", J. of Rad. Res. 43 Suppl(S):S149-52. doi: 10.1269/jrr.43.S149

Sylvester, C. B. et al. (2018), “Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer”, Frontiers in cardiovascular medicine, Vol. 5, Frontiers, https://doi.org/10.3389/fcvm.2018.00005

Terato, H. & H. Ide (2005), "Clustered DNA damage induced by heavy ion particles.", Biol. Sci. Sp. 18(4):206-215. doi:10.2187/bss.18.206.

Ungvari, Z. et al. (2013), “Ionizing radiation promotes the acquisition of a senescence-associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: role of increased DNA damage and decreased DNA repair capacity in microvascular radiosensitivity”, The journals of gerontology. Series A, Biological sciences and medical sciences, Vol. 68/12, Oxford University Press, Oxford, https://doi.org/10.1093/gerona/glt057

Valentin, J.D.J (1998), "Chapter 1. Ann ICRP.", 28(4):5-7. doi:10.1016/S0146-6453(00)00002-6. http://www.ncbi.nlm.nih.gov/pubmed/10882804.

Venkatesh, P. et al. (2016), "Effect of chromatin structure on the extent and distribution of DNA double strand breaks produced by ionizing radiation; comparative study of hESC and differentiated cells lines.", Int J. Mol. Sci. 17(1). doi:10.3390/ijms17010058.

Wang, et al. (2021), “Ionizing Radiation-Induced Brain Cell Aging and the Potential Underlying Molecular Mechanisms.”, Cells. 10(12):3570. doi: 10.3390/cells10123570

Ward, J. F. (1988), "DNA Damage Produced by Ionizing Radiation in Mammalian Cells: Identities, Mechanisms of Formation, and Reparability.", Prog. Nucleic Acid Res. Mol. Biol. 35(C):95-125. doi:10.1016/S0079-6603(08)60611-X.

Wolf, N. et al. (2008), “Radiation cataracts: Mechanisms involved in their long delayed occurrence but then rapid progression”, Molecular vision, Vol. 14/34-35, Molecular Vision, Atlanta, pp. 274-285

Wu, L.J. et al. (1999), "Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells.", Proc. Natl. Acad. Sci. 96(9):4959-4964. doi:10.1073/pnas.96.9.4959.

Yamaguchi, H. et al. (2005), "Estimation of Yields of OH Radicals in Water Irradiated by Ionizing Radiation.", J. of Rad. Res. 46(3):333-41. doi: 10.1269/jrr.46.333.

Zhang, L. et al. (2017), “The inhibitory effect of minocycline on radiation-induced neuronal apoptosis via AMPKα1 signaling-mediated autophagy.”, Sci Rep.7(1):16373. doi: 10.1038/s41598-017-16693-8.
## AOPs Referencing Relationship

<table><thead><tr><th>AOP Name</th><th>Adjacency</th><th>Weight of Evidence</th><th>Quantitative Understanding</th></tr></thead><tbody><tr><td>AOP: 470 - Deposition of energy leads to abnormal vascular remodeling</td><td>adjacent</td><td>High</td><td>Moderate</td></tr><tr><td>AOP: 483 - Deposition of Energy Leading to Learning and Memory Impairment</td><td>adjacent</td><td>Moderate</td><td>Low</td></tr></tbody></table>

## Evidence Supporting Applicability of this Relationship

Evidence for this relationship is predominantly from studies using rat- and mouse-derived cells, with some in vivo evidence in mice and rats. There is in vivo evidence in male animals, but no in vivo studies specify the use of female animals. In vivo evidence is from adult models.

### Taxonomic Applicability

<table><thead><tr><th>Term</th><th>Scientific Term</th><th>Evidence</th><th>Link</th></tr></thead><tbody><tr><td>human</td><td>Homo sapiens</td><td>Low</td><td>NCBI</td></tr><tr><td>mouse</td><td>Mus musculus</td><td>Moderate</td><td>NCBI</td></tr><tr><td>rat</td><td>Rattus norvegicus</td><td>Moderate</td><td>NCBI</td></tr></tbody></table>

### Life Stage Applicability

<table><thead><tr><th>Term</th><th>Evidence</th></tr></thead><tbody><tr><td>Juvenile</td><td>Low</td></tr><tr><td>Adult</td><td>Moderate</td></tr></tbody></table>

### Sex Applicability

<table><thead><tr><th>Sex</th><th>Evidence</th></tr></thead><tbody><tr><td>Male</td><td>Moderate</td></tr><tr><td>Female</td><td>Low</td></tr></tbody></table>

## Key Event Relationship Description

DNA strand breaks can lead to altered signaling of various pathways through the DNA damage response. DNA strand breaks, which are a form of DNA damage, can induce ataxia telangiectasia mutated (ATM) and ATM/RAD3-related (ATR), two phosphoinositide 3-kinase (PI3K)-related serine/threonine kinases (PIKKs) (Abner and McKinnon, 2004; Lee and McKinnon, 2007; Nagane et al., 2021; Sylvester et al., 2018; Thadathil et al., 2019; Wang et al., 2020; Wang et al., 2017). Following DNA strand breaks, cellular DNA damage response signaling can phosphorylate downstream proteins and activate several transcription factors and pathways (Wang et al., 2017). Spontaneous DNA strand breaks from endogenous sources will induce signaling as a normal response to facilitate DNA repair. However, excessive DNA damage induced by a stressor will result in increased activation of these pathways and subsequent harmful downstream effects. Stress response signaling pathways induced by DNA strand breaks include p53/p21 (Abner and McKinnon, 2004; Baselet et al., 2018; Lee and McKinnon, 2007; Nagane et al., 2021; Sylvester et al., 2018; Thadathil et al., 2019; Wang et al., 2020; Wang et al., 2017), caspase (Abner and McKinnon, 2004; Baselet et al., 2019; Wang et al., 2020; Wang et al., 2016) and mitogen-activated protein kinase (MAPK) family pathways (Ghahremani et al., 2002; Nagane et al., 2021).

## Evidence Supporting this KER

Overall weight of evidence: Moderate

### Biological Plausibility
There is strong evidence supporting the link between DNA strand breaks leading to altered stress response signaling. Single strand breaks (SSBs) or double strand breaks (DSBs) in DNA from both endogenous and exogenous sources can induce the DNA damage response, which can result in the induction of various signaling pathways (Baselet et al., 2019). DNA strand breaks are well known to lead to the activation of ATM and ATR as part of the normal DNA damage response (Abner and McKinnon, 2004; Baselet et al., 2019; Lee and McKinnon, 2007; Nagane et al., 2021; Sylvester et al., 2018; Thadathil et al., 2019; Wang et al., 2020; Wang et al., 2017; Wang et al., 2016). While ATM tends to be recruited to DSBs, ATR is recruited by many types of DNA damage including both DSBs and SSBs (Maréchal and Zou, 2013; Wang et al., 2017). Following a DNA DSB, the Mre11-Rad50-Nbs1 (MRN) complex senses and directly binds to the DNA ends at the site of the break, which subsequently activates ATM (Lee and McKinnon, 2007; Maréchal and Zou, 2013). Following a DNA SSB, resection of the damaged strand by apurinic/apyrimidinic endonuclease (APE)1/APE2 is followed by coating the single-stranded DNA with replication protein A (RPA), where the recruitment of the ATR- ATR interacting protein (ATRIP) complex and the activation of ATR occurs (Caldecott, 2022; Maréchal and Zou, 2013).

ATM and ATR can phosphorylate over 700 proteins (Nagane et al., 2021), and phosphorylation of key signaling proteins by ATM/ATR will alter signaling in their respective pathways. High levels of DNA strand breaks induced by exogenous stressors will enhance ATM/ATR activation and subsequently further activate downstream signaling, leading to downstream consequences. The extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK subfamily pathways can be phosphorylated and activated by ATM/ATR (Ghahremani et al., 2002; Nagane et al., 2021). Additionally, ATM/ATR can phosphorylate p53 on serine 15 to enhance the stability of p53, leading to activation of the p53 pathway and changes in the transcriptional activity of p53 (Abner and McKinnon, 2004; Baselet et al., 2019; Lee and McKinnon, 2007; Nagane et al., 2021; Sylvester et al., 2018; Thadathil et al., 2019; Wang et al., 2020; Wang et al., 2017; Wang et al., 2016). The apoptosis pathway downstream of p53 can also be activated by DNA strand breaks (Abner and McKinnon, 2004; Baselet et al., 2019; Lee and McKinnon, 2007; Thadathil et al., 2019; Wang et al., 2020).

Empirical Evidence

Evidence for this relationship was collected from studies using in vivo models of mice and rats as well as in vitro models of cells derived from human, mice or rats. The stressors used to support this relationship include 137Cs gamma rays and X rays. Markers of DNA strand breaks in this KER include p53 binding protein 1 (53BP1), phosphorylation of H2AX (γ-H2AX), phosphorylation of ATR (p-ATR) and phosphorylation of ATM (p-ATM). Altered signaling was measured mostly by the protein expression of the p53/p21 and apoptosis pathways.

Dose Concordance

A few studies have indicated a dose concordance between the increase in DNA strand breaks and altered stress response signaling. X-ray irradiation of rat cortical neurons showed increased DNA damage markers, γ-H2AX, p-ATM and p-ATR and increased levels of signaling proteins, including p21, p-p53 and cleaved caspase 3 at both doses of 8 and 32 Gy (Sabirzhanov et al., 2020). 137Cs gamma irradiated cerebromicrovascular endothelial cells (CMVECs) and rat hippocampal neurons showed increased DNA strand breaks, measured by comet assay, at 2-10 Gy, and increased caspase 3/7 activity at 2, 4 and 6 Gy (Ungvari et al., 2013).

Time Concordance

Many studies demonstrate that DNA strand breaks occur before altered signaling in a time course. Although both KEs can occur quickly, Gionchiglia et al. (2021) showed in mice that γ-H2AX and p53BP1 foci were increased as early as 15 minutes after 10 Gy of X-ray irradiation while cleaved caspase 3 did not increase until 30 minutes after irradiation. In HT22 hippocampal neurons irradiated with 12 Gy of X-rays, γ-H2AX and p-ATM were increased at 30 minutes post-irradiation while p53 was increased after 1 h and caspase 3 was increased after 48 h (Zhang et al., 2017). Similarly, rat cortical neurons irradiated with 8 Gy of X-rays showed increased p-ATM, γ-H2AX and p-ATR after
30 minutes, while p-p53, p21 and cleaved caspase 3 did not increase until 3 or 6 h post-irradiation (Sabirzhanov et al., 2020). Multiple studies using human- and rat-derived endothelial cells irradiated with 4 Gy of 137Cs gamma rays show increased DNA strand breaks at 1 h post-irradiation, with altered signaling to p53 and p21 at 6 h and to caspase 3/7 at 18 h post-irradiation (Kim et al., 2014; Park et al., 2022; Ungvari et al., 2013). In a longer-term study irradiating human lung microvascular endothelial cells (HMVEC-L) with 15 Gy of X-rays, increased DNA strand breaks were observed at 14 days post-irradiation, while altered signaling in the p53 pathway was observed at 21 days post-irradiation (Lafargue et al., 2017).

### Incidence concordance

A few studies have demonstrated an incidence concordance between DNA strand breaks and altered signaling at equivalent doses. Following X-ray irradiation of mice, DNA damage markers, γ-H2AX and p53BP1, increased by 10, 15 and 5-fold in different region of the brain, while cleaved caspase 3 signaling molecule increased by 1.4 and 2.6-fold (Gionchiglia et al., 2021). Gamma ray irradiation of Wistar rats showed a 6-fold increase in DNA damage marker compared to a 0.2-fold decrease in (B-cell lymphoma 2) Bcl-2 and a 2- to 4-fold increase in signaling proteins p53, Bcl-2-associated protein X (Bax) and caspase 3/8/9 (El-Missiry et al., 2018).

### Essentiality

Some studies show that preventing an increase in DNA strand breaks will restore signaling. Treatment with mesenchymal stem cell- conditioned medium (MSC-CM) reduced γ-H2AX, decreased the levels of p53, Bax, cleaved caspase 3 and increased the levels of Bcl-2 in HT22 cells irradiated with 10 Gy of X-rays (Huang et al., 2021). The inhibition of microRNA (miR)-711 decreased levels of DNA damage markers, p-ATM, p-ATR and γ-H2AX, and decreased signaling molecules including p-p53, p21 and cleaved caspase 3 (Sabirzhanov et al., 2020).

### Known modulating factors

<table><thead><tr><td>Modulating Factor</td><td>Details</td><td>Effects on the KER</td><td>References</td></tr></thead><tbody><tr><td>Nitroxides</td><td>Increased concentration</td><td>Decreased DNA strand breaks.</td><td>DeGraff et al., 1992; Citrin & Mitchel, 2014</td></tr><tr><td>5-fluorouracil</td><td>Increased concentration</td><td>Increased DNA strand breaks.</td><td>De Angelis et al., 2006; Citrin & Mitchel, 2014</td></tr><tr><td>Thiols</td><td>Increased concentration</td><td>Decreased DNA strand breaks.</td><td>Milligan et al., 1995; Citrin & Mitchel, 2014</td></tr><tr><td>Cisplatin</td><td>Increased concentration</td><td>Decreased DNA break repair.</td><td>Sears & Turchi; Citrin & Mitchel, 2014</td></tr></tbody></table>

### Quantitative Understanding of the Linkage

The tables below provide some representative examples of quantitative linkages between the two key events. All data that is represented is statistically significant unless otherwise indicated.

### Dose Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Sabirzhanov et al., 2020</td><td>In vitro. Rat cortical neurons were exposed to 2, 8 and 32 Gy X-rays. DNA damage was determined by γ-H2AX staining and western blot analysis of p-ATM and p-ATR. Altered signaling was determined by levels of p-p53, p21, cleaved caspase 3, measured by Western blot.</td><td>Irradiated primary cortical neurons showed increased γ-H2AX by 30-fold at both 8 and 32 Gy but not at 2 Gy. p-ATM was increased at all doses, increasing about 15-fold at 8 and 32 Gy. Signaling molecules including p-p53, p21, and cleaved caspase 3 were increased at all doses.</td></tr><tr><td>Ungvari et al., 2013</td><td>In vitro. CMVECs and rat hippocampal neurons were irradiated with 137Cs gamma rays. DNA strand breaks were assessed with the comet assay. Caspase 3/7 activity was determined by an assay kit.</td><td>DNA damage increased at all doses (2-10 Gy). In the control, less than 5% of DNA content was in the tail while by 6 Gy 35% of the DNA content was in the tail in CMVECs and 25% was in the tail in neurons. In CMVECs, 2, 4, and 6 Gy increased caspase 3/7 activity 5- to 6-fold.</td></tr></tbody></table>

### Time Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead></table>
<table><tr><td>Zhang et al., 2017</td><td>In vitro. HT22 cells were irradiated with 12 Gy of X-rays (1.16 Gy/min). p-ATM, γ-H2AX, cleaved caspase 3 and p53 were measured with Western blot.</td><td>p-ATM and γ-H2AX were increased 4.4-fold and 3.2-fold, respectively, 30 min after 12 Gy. p53 was increased 4.6-fold at 1 h post-irradiation. A 9-fold increase in cleaved caspase 3 was observed 48 h post-irradiation.</td></tr><tr><td>Gionchiglia et al., 2021</td><td>In vivo. CD1 and B6/129 mice were irradiated with 10 Gy of X-rays. γ-H2AX and 53BP1 foci were quantified with immunofluorescence in neurons. Cleaved caspase 3 positive neurons were measured with immunofluorescence.</td><td>At both 15 and 30 min post-irradiation, γ-H2AX and p53BP1 foci increased. However, cleaved caspase 3 increased at 30 min but not at 15 min.</td></tr><tr><td>Sabirzhanov et al., 2020</td><td>In vitro. Rat cortical neurons were exposed to 2, 8 and 32 Gy X-rays. DNA damage was determined by γ-H2AX staining and western blot analysis of p-ATM and p-ATR. Altered signaling was determined by levels of p-p53, p21, cleaved caspase 3, measured by Western blot.</td><td>DNA damage occurred as early as 30 min post 8 Gy irradiation, indicated by increased p-ATM, γ-H2AX and p-ATR. Signaling molecules p-p53, p21 and cleaved caspase 3 increased at 3 or 6h post-irradiation.</td></tr><tr><td>Park et al., 2022</td><td>In vitro. Human aortic endothelial cells (HAECs) were irradiated with 4 Gy of 137Cs gamma rays (3.5 Gy/min). γ-H2AX was measured with western blot. p-ATM and 53BP1 were determined with immunofluorescence. p-p53 and p21 were measured with Western blot.</td><td>γ-H2AX, p-ATM, and 53BP1 increased at 1 h post-irradiation, while p-p53 and p21 were increased at 6 h post-irradiation.</td></tr><tr><td>Kim et al., 2014</td><td>In vitro. Human umbilical vein endothelial cells (HUVECs) were irradiated with 4 Gy 137Cs gamma rays. DNA damage was determined by γ-H2AX. p21 and p53 were measured by Western blot.</td><td>γ-H2AX foci greatly increased at 1 and 6 h post-irradiation, while p-p53 and p21 were increased at 6 h post-irradiation.</td></tr><tr><td>Ungvari et al., 2013</td><td>In vitro. CMVECs and rat hippocampal neurons were irradiated with 2-6 Gy of 137Cs gamma rays. DNA strand breaks were assessed with the comment assay. Caspase 3/7 activity was determined by an assay kit.</td><td>DNA damage in neurons and CMVECs increased at 1 h post-irradiation, while caspase 3/7 activity increased the greatest at 18 h post-irradiation in CMVECs.</td></tr><tr><td>Lafargue et al., 2017</td><td>In vitro. HMVEC-L were irradiated with 15 Gy of X-rays. γ-H2AX foci were assessed with immunofluorescence. p-ATM and ATM were assessed with Western blot. Signaling proteins including p53, p21 and p16 were assessed with Western blot.</td><td>Without irradiation, most cells had 0 or 1 γ-H2AX foci, while 14 days after 15 Gy, most cells had 2-6 γ-H2AX foci. The ratio of p-ATM/ATM was also increased 14 days after 15 Gy, p53, p21, and p16 were all increased at 21 days after 15 Gy.</td></tr></table>

Incidence Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>El-Missiry et al., 2018</td><td>In vivo. Wistar rats were irradiated with 4 Gy of 137Cs gamma rays (0.695 cGy/s). DNA damage was assessed with a comet assay. Multiple signaling proteins were assessed with assay kits.</td><td>The tail moment increased 6-fold while signaling proteins including p53, Bax, and caspases 3/8/9 increased 2- to 4-fold, and Bcl-2 decreased 0.2-fold.</td></tr><tr><td>Gionchiglia et al., 2021</td><td>In vivo. CD1 and B6/129 mice were irradiated with 10 Gy of X-rays. γ-H2AX and 53BP1 foci were quantified with immunofluorescence. Cleaved caspase 3 positive cells were measured with immunofluorescence.</td><td>γ-H2AX and p53BP1 foci increased about 10-fold in the forebrain and cerebral cortex, about 15-fold in the hippocampus and about 5-fold in the subventricular zone (SVZ)/ rostral migratory stream (RMS)/ olfactory bulb (OB). Cleaved caspase 3 increased 1.4-fold in the cerebral cortex and hippocampus and 2.6-fold in the SVZ/RMS/OB.</td></tr></tbody></table>

Known Modulating Factors

<table><thead><tr><td>Modulating Factor</td><td>Details</td><td>Effects on the KER</td><td>References</td></tr></thead><tbody><tr><td>Nitroxides</td><td>Increased concentration</td><td>Decreased DNA strand breaks.</td><td>DeGraff et al., 1992; Citrin &amp; Mitchel, 2014</td></tr><tr><td>5-fluorouracil</td><td>Increased concentration</td><td>Increased DNA strand breaks.</td><td>De Angelis et al., 2006; Citrin &amp; Mitchel, 2014</td></tr><tr><td>Thiols</td><td>Increased concentration</td><td>Decreased DNA strand breaks.</td><td>Milligan et al., 1995; Citrin &amp; Mitchel, 2014</td></tr><tr><td>Cisplatin</td><td>Increased concentration</td><td>Decreased DNA break repair.</td><td>Sears &amp; Turchi; Citrin &amp; Mitchel, 2014</td></tr></tbody></table>
Agrawala, P.K. et al. (2008), "Induction and repairability of DNA damage caused by ultrasoft X-rays: Role of core events.", Int. J. Radiat. Biol., 84(12):1093-1103. doi:10.1080/09553000802478083.

Ahmadi, M. et al. (2021), "Early responses to low-dose ionizing radiation in cellular lens epithelial models", Radiation research, Vol.197/1, Radiation Research Society, United States, https://doi.org/10.1667/RADE-20-00284.1

Ainsbury, E. A. et al. (2016), "Ionizing radiation induced cataracts: Recent biological and mechanistic developments and perspectives for future research", Mutation research. Reviews in mutation research, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.07.010

Alexander, J. L. and Orr-Weaver, T. L. (2016), "Replication fork instability and the consequences of fork collisions from replication", Genes & Development, Vol. 30/20, Cold Spring Harbor Laboratory Press, https://doi.org/10.1101/gad.288142.116

Allen, C. H. et al. (2018), "Raman micro-spectroscopy analysis of human lens epithelial cells exposed to a low-dose-range of ionizing radiation", Physics in medicine & biology, Vol. 63/2, IOP Publishing, Bristol, https://doi.org/10.1088/1361-6560/aaa176

Antonelli, A.F. et al. (2015), "Induction and Repair of DNA DSB as Revealed by H2AX Phosphorylation Foci in Human Fibroblasts Exposed to Low- and High-LET Radiation: Relationship with Early and Delayed Reproductive Cell Death", Radiat. Res. 183(4):417-31, doi:10.1667/RR13855.1.

Acharya, M. et al. (2010), "Consequences of ionizing radiation-induced damage in human neural stem cells", Free Radical Biology and Medicine. 49(12):1846-1855, doi:10.1016/j.freeradbiomed.2010.08.021.

Asaithamby, A. et al. (2008), "Repair of HZE-Particle-Induced DNA Double-Strand Breaks in Normal Human Fibroblasts.", Radiat Res. 169(4):437-446. doi:10.1667/RR1165.1.

Aufderheide, E. et al. (1987), "Heavy ion effects on cellular DNA: Strand break induction and repair in cultured diploid lens epithelial cells", International journal of radiation biology and related studies in physics, chemistry and medicine, Vol. 51/5, Taylor & Francis, London, https://doi.org/10.1080/09553008714551071

Bannik, K. et al. (2013), "Are mouse lens epithelial cells more sensitive to γ-irradiation than lymphocytes?", Radiation and environmental biophysics, Vol. 52/2, Springer-Verlag, Berlin/Heidelberg, https://doi.org/10.1007/s00411-012-0451-8

Bains, S. K. et al. (2019), "Effects of ionizing radiation on telomere length and telomerase activity in cultured human lens epithelium cells", International journal of radiation biology, Vol. 95/1, Taylor & Francis, Abingdon, https://doi.org/10.1080/09553002.2018.1466066

Barazzuol, L et al. (2017), "A coordinated DNA damage response promotes adult quiescent neural stem cell activation. PLOS Biology, 15(5). https://doi.org/10.1371/journal.pbio.2001264

Barnard, S. G. R. et al. (2018), "Dotting the eyes: mouse strain dependency of the lens epithelium to low dose radiation-induced DNA damage", International journal of radiation biology, Vol. 94/12, Taylor & Francis, Abingdon, https://doi.org/10.1080/09553002.2018.1532609
Barnard, S. G. R. et al. (2019), "Inverse dose-rate effect of ionising radiation on residual 53BP1 foci in the eye lens", Scientific Reports, Vol. 9/1, Nature Publishing Group, England, https://doi.org/10.1038/s41598-019-46893-3

Barnard, S. G. R. et al. (2022), "Radiation-induced DNA damage and repair in lens epithelial cells of both Ptch1 (+/-) and Ercc2 (+/-) mutated mice", Radiation Research, Vol. 197/1, Radiation Research Society, United States, https://doi.org/10.1667/RADE-20-00264.1

Baselet, B. et al. (2019), "Pathological effects of ionizing radiation: endothelial activation and dysfunction", Cellular and molecular life sciences, Vol. 76/4, Springer Nature, https://doi.org/10.1007/s00018-018-2956-z

Baselet, B. et al. (2017), "Functional Gene Analysis Reveals Cell Cycle Changes and Inflammation in Endothelial Cells Irradiated with a Single X-ray Dose", Frontiers in pharmacology, Vol. 8, Frontiers, https://doi.org/10.3389/fphar.2017.00213

Baumstark-Khan, C., J. Heilmann, and H. Rink (2003), ‘Induction and repair of DNA strand breaks in bovine lens epithelial cells after high LET irradiation”, Advances in space research, Vol. 31/6, Elsevier Ltd, England, https://doi.org/10.1016/S0273-1177(03)00095-4

Beels, L. et al. (2009), "g-H2AX Foci as a Biomarker for Patient X-Ray Exposure in Pediatric Cardiac Catheterization", Are We Underestimating Radiation Risks?": 1903–1909. doi:10.1161/CIRCULATIONAHA.109.880385.

Belli M, Cherunbini R, Vecchia MD, Dini V, Moschini G, Signoretti C, Simon G, Tabocchini MA, Tiveron P. 2000. DNA DSB induction and rejoining in V79 cells irradiated with light ions: a constant field gel electrophoresis study. Int J Radiat Biol. 76(8):1095-1104.

Brooks, A.L., D.G. Hoel & R.J. Preston (2016), "The role of dose rate in radiation cancer risk: evaluating the effect of dose rate at the molecular, cellular and tissue levels using key events in critical pathways following exposure to low LET radiation.", Int. J. Radiat. Biol. 92(8):405-426. doi:10.1080/09553002.2016.1186301.

Bucolo, C. et al. (1994), “The effect of ganglioside on oxidation-induced permeability changes in lens and in epithelial cells of lens and retina”, Experimental eye research, Vol. 58/6, Elsevier Ltd, London, https://doi.org/10.1006/exer.1994.1067

Cabrera et al. (2011), “Antioxidants and the integrity of ocular tissues”, in Veterinary medicine international, SAGE-Hindawi Access to Research, United States. DOI: 10.4061/2011/905153

Cadet, J. et al. (2012), “Oxidatively generated complex DNA damage: tandem and clustered lesions”, Cancer letters, Vol. 327/1, Elsevier Ireland Ltd, Ireland. https://doi.org/10.1016/j.canlet.2012.04.005

Cannan, W.J. & D.S. Pederson (2016), "Mechanisms and Consequences of Double-Strand DNA Break Formation in Chromatin.", J. Cell Physiol. 231(1):3-14. doi:10.1002/jcp.25048.

Cencer, C. S. et al. (2018), “PARP-1/PAR activity in cultured human lens epithelial cells exposed to two levels of UVB light”, Photochemistry and photobiology, Vol. 94/1, Wiley, Hoboken, https://doi.org/10.1111/php.12814

Chadwick, K.H., (2017), Towards a new dose and dose-rate effectiveness factor (DDREF)? Some comments., J Radiol Prot., 37:422-433. doi: 10.1088/1361-6498/aa6722.
Charlton, D.E., H. Nikjoo & J.L. Humm (1989), "Calculation of initial yields of single- and double-strand breaks in cell nuclei from electrons, protons and alpha particles.", Int. J. Rad. Biol., 53(3):353-365, DOI: 10.1080/09553008814552501

Christensen, D.M. (2014), "Management of Ionizing Radiation Injuries and Illnesses, Part 3: Radiobiology and Health Effects of Ionizing Radiation.", 114(7):556–565. doi:10.7556/jaoa.2014.109.

Citrin, D.E. & J.B. Mitchel (2014), "Public Access NIH Public Access.", 71(2):233–236. doi:10.1038/mp.2011.182.doi.

Dalke, C. et al. (2018), “Lifetime study in mice after acute low-dose ionizing radiation: a multifactorial study with special focus on cataract risk”, Radiation and environmental biophysics, Vol. 57/2, Springer Berlin Heidelberg, Berling/Heidelberg, https://doi.org/10.1007/s00411-017-0728-z

Day, T.K. et al. (2007), "Adaptive Response for Chromosomal Inversions in pKZ1 Mouse Prostate Induced by Low Doses of X Radiation Delivered after a High Dose.", Radiat Res. 167(6):682–692. doi:10.1667/rr0764.1.

De Angelis, P. M. et al. (2006), “Cellular response to 5-fluorouracil (5-FU) in 5-FU-resistant colon cancer cell lines during treatment and recovery”, Molecular Cancer, Vol. 5/20, BioMed Central, https://doi.org/10.1186/1476-4598-5-20

DeGraff, W. G. et al. (1992), “Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage”, Free Radical Biology and Medicine, Vol. 13/5, Elsevier, https://doi.org/10.1016/0891-5849(92)90142-4

Desouky, O., N. Ding & G. Zhou (2015), "ScienceDirect Targeted and non-targeted effects of ionizing radiation.", J. Radiat. Res. Appl. Sci. 8(2):247–254. doi:10.1016/j.jrras.2015.03.003.

Dong et al. (2015), “Relationship between irradiation-induced neuro-inflammatory environments and impaired cognitive function in the developing brain of Mice. International Journal of Radiation Biology, 91(3):224–239. https://doi.org/10.3109/09553002.2014.988895

Dong, X. et al. (2014), “NEMO modulates radiation-induced endothelial senescence of human umbilical veins through NF-κB signal pathway”, Radiation Research, Vol. 183/1, BioOne, https://doi.org/10.1667/RR13682.1

Dubrova, Y.E. & M.A. Plumb (2002), "Ionising radiation and mutation induction at mouse minisatellite loci The story of the two generations", Mutat. Res. 499(2):143–150. EPRI (2014), Epidemiology and mechanistic effects of radiation on the lens of the eye: Review and scientific appraisal of the literature, EPRI, California

Falk, M., E. Lukášová & S. Kozubek (2008), "Chromatin structure influences the sensitivity of DNA to γ-radiation.", Biochim. Biophys. Acta. - Mol. Cell. Res. 1783(12):2398–2414. doi:10.1016/j.bbamcr.2008.07.010.

Feinendegen, L.E. (2005), "UKRC 2004 debate Evidence for beneficial low level radiation effects and radiation hormesis. Radiology.", 78:3–7. doi:10.1259/bjr/63353075. Feinendegen, L.E., M. Pollycove & R.D. Neumann (2007), "Whole-body responses to low-level radiation exposure: New concepts in mammalian radiobiology.", Exp. Hematol. 35(4 SUPPL.):37–46. doi:10.1016/j.exphem.2007.01.011.

Flegal, M. et al. (2015), "Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and γ-Radiation.", (July):1–9. doi:10.3791/52912.
Foray, N., M. Bourguignon and N. Hamada (2016), "Individual response to ionizing radiation", Mutation research. Reviews in mutation research, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.09.001

Franken NAP, Hovingh S, Cate RT, Krawczyk P, Stap J, Hoebe R, Aten J, Barendsen GW. 2012. Relative biological effectiveness of high linear energy transfer alpha-particles for the induction of DNA-double-strand breaks, chromosome aberrations and reproductive cell death in SW-1573 lung tumour cells. Oncol reports. 27:769-774.

Frankenberg D, Brede HJ, Schrewe UJ, Steinmetz C, Frankenberg-Scwager M, Kasten G, Pralle E. 1999. Induction of DNA Double-Strand Breaks by 1H and 4He Ions in Primary Human Skin Fibroblasts in the LET range of 8 to 124 keV/µm. Radiat Res. 151:540-549.

Geisel, D. et al. (2012), "DNA double-strand breaks as potential indicators for the biological effects of ionising radiation exposure from cardiac CT and conventional coronary angiography: a randomised, controlled study", European Radiology, Vol. 22/8, Springer Nature, https://doi.org/10.1007/s00330-012-2426-1

Gionchiglia, N. et al. (2021), "Association of Caspase 3 Activation and H2AX γ Phosphorylation in the Aging Brain: Studies on Untreated and Irradiated Mice," Biomedicines. 9(9):1166. doi: 10.3390/biomedicines9091166.

Goodhead, D.T. (2006), "Energy deposition stochastics and track structure: What about the target?", Radiat. Prot. Dosimetry. 122(1-4):3-15. doi:10.1093/rpd/ncl498.

Grudzenski, S. et al. (2010), "Inducible response required for repair of low-dose radiation damage in human fibroblasts.", Proc. Natl. Acad. Sci. USA. 107(32): 14205-14210, doi:10.1073/pnas.1002213107.

Hada, M. & A.G. Georgakilas (2008), "Formation of Clustered DNA Damage after High-LET Irradiation: A Review.", J. Radiat. Res., 49(3):203-210. doi:10.1269/jrr.07123.

Hamada, N. et al. (2006), "Histone H2AX phosphorylation in normal human cells irradiated with focused ultrasoft X rays: evidence for chromatin movement during repair", Radiation Research, Vol. 166/1, Radiation Research Society, United States, https://doi.org/10.1667/RR3577.1

Hamada, N. (2014), "What are the intracellular targets and intratissue target cells for radiation effects?", Radiation research, Vol. 181/1, The Radiation Research Society, Lawrence, https://doi.org/10.1667/RR13505.1

Hamada, N. and T. Sato (2016), "Cataractogenesis following high-LET radiation exposure", Mutation Research. Reviews in mutation research, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.08.005

Hamada, N. (2017a), "Ionizing radiation sensitivity of the ocular lens and its dose rate dependence", International journal of radiation biology, Vol. 93/10, Taylor & Francis, Abingdon, https://doi.org/10.1080/09553002.2016.1266407

Hamada, N. (2017b), "Ionizing radiation response of primary normal human lens epithelial cells", PloS ONE, Vol. 12/7, Public Library Science, San Francisco, https://doi.org/10.1371/journal.pone.0181530

Havas, M. (2017), "When theory and observation collide: Can non-ionizing radiation cause cancer?", Environmental pollution, Vol. 221, Elsevier Ltd, England. https://doi.org/10.1016/j.envpol.2016.10.018
Hightower, K. R. (1995), "The role of the lens epithelium in development of UV cataract", Current eye research, Vol. 14/1, Informal UK Ltd, Philadelphia, https://doi.org/10.3109/02713689508999916

Iliakis, G., T. Murmann & A. Soni (2015), "Alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations.", Mutat. Res. - Genet. Toxicol. Environ. Mutagen. 793:166-175. doi:10.1016/j.mrgentox.2015.07.001.

Joiner, M. (2009), "Basic Clinical Radiobiology", Edited by. [1] P.J. Sadler, Next-Generation Met Anticancer Complexes Multitargeting via Redox Modul Inorg Chem 52 21.:375. doi:10.1201/b13224.

Jorge, S.-G. et al. (2012), "Evidence of DNA double strand breaks formation in Escherichia coli bacteria exposed to alpha particles of different LET assessed by the SOS response.", Appl. Radiat. Isot. 71(SUPPL.):66-70. doi:10.1016/j.apradiso.2012.05.007.

Kadhim, M.A., M.A. Hill & S.R. Moore, (2006), "Genomic instability and the role of radiation quality.", Radiat. Prot. Dosimetry. 122(1-4):221-227. doi:10.1093/rpd/ncl445. Khanna, K.K. & S.P. Jackson (2001), "DNA double-strand breaks: signaling, repair and the cancer connection.", Nature Genetics. 27(3):247-54. doi:10.1038/85798.

Kim, K. S. et al. (2014), "Characterization of DNA damage-induced cellular senescence by ionizing radiation in endothelial cells", International Journal of Radiation Biology, Vol. 90/1, Informa, London, https://doi.org/10.3109/09553002.2014.859763

Kim, J. N. and B. M. Lee (2007), "Risk factors, health risks, and risk management for aircraft personnel and frequent flyers", Journal of toxicology and environmental health. Part B, Critical reviews, Vol. 10/3, Taylor & Francis Group, Philadelphia, https://doi.org/10.1080/10937400600882103

Kleiman, N. J., R. Wang and A. Spector (1990), "Ultraviolet light induced DNA damage and repair in bovine lens epithelial cells", Current eye research, Vol. 9/12, Informa UK Ltd, Oxford, https://doi.org/10.3109/02713689009003475

Kozbenko, T. et al. (2022), "Deploying elements of scoping review methods for adverse outcome pathway development: a space travel case example", International Journal of Radiation Biology, 1-12. https://doi.org/10.1080/09553002.2022.2110306

Kuefner, M.A. et al. (2009), "DNA Double-Strand Breaks and Their Repair in Blood Lymphocytes of Patients Undergoing Angiographic Procedures.", Investigative radiology. 44(8):440-6. doi:10.1097/RLI.0b013e3181a654a5.

Kuefner, M.A. et al. (2015), "Chemoprevention of Radiation-Induced DNA Double-Strand Breaks with Antioxidants.", Curr Radiol Rep (2015) 3: 81. https://doi.org/10.1007/s40134-014-0081-9

Kuhne, M., G. Urban & M. Lo, (2005), "DNA Double-Strand Break Misrejoining after Exposure of Primary Human Fibroblasts to CK Characteristic X Rays, 29 kVp X Rays and Co γ-Rays.", Radiation Research. 164(5):669-676. doi:10.1667/RR3461.1.

de Lara, C.M. et al. (2001), "Dependence of the Yield of DNA Double-Strand Breaks in Chinese Hamster V79-4 Cells on the Photon Energy of Ultrasoft X Rays.", Radiation Research. 155(3):440-8. doi:10.1667/0033-7587(2001)155[0440:DOTYOD]2.0.CO;2.

Liao, J. et al. (2011), "Anti-UVC irradiation and metal chelation properties of 6-benzoyl-5,7-dihydroxy-4-phenyl-chromen-2-one: An implications for anti-cataract agent", International journal of molecular sciences, Vol. 12/10, MDPI, Basel. https://doi.org/10.3390/ijms12107059
Lipman, R. M., B. J. Tripathi, R. C. Tripathi (1998), "Cataracts induced by microwave and ionizing radiation", Survey of ophthalmology, Vol. 33/3, Elsevier Inc, United States, https://doi.org/10.1016/0039-6257(88)90088-4

Lomax, M.E., L.K. Folkes & P.O. Neill (2013). "Biological Consequences of Radiation-induced DNA Damage: Relevance to Radiotherapy", Statement of Search Strategies Used and Sources of Information Why Radiation Damage is More Effective than Endogenous Damage at Killing Cells Ionising Radiation-induced Do. 25:578-585. doi:10.1016/j.clon.2013.06.007.

Long, A. C., C. M. H. Colitz, and J. A. Bomser (2001), "Apoptotic and necrotic mechanisms of stress-induced human lens epithelial cell death", Experimental biology and medicine, SAGE Publications, London, https://doi.org/10.1177/153537020422901012

Lorat, Y. et al. (2015), "Nanoscale analysis of clustered DNA damage after high-LET irradiation by quantitative electron microscopy – The heavy burden to repair.", DNA Repair (Amst). 28:93-106. doi:10.1016/j.dnarep.2015.01.007.

Maier, P. et al. (2016), "Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization.", Int. J. Mol. Sci., 14;17(1), pii:E102, doi:10.3390/ijms17010102.

Markiewicz, E. et al. (2015), "Nonlinear ionizing radiation-induced changes in eye lens cell proliferation, cyclin D1 expression and lens shape", Open biology, Vol. 5/4, Royal society, London, https://doi.org/10.1098/rsob.150011

Milligan, J. R. et al. (1995), « DNA repair by thiols in air shows two radicals make a double-strand break", Radiation Research, Vol 143/3, pp. 273-280

Moore, S., F.K.T. Stanley & A.A. Goodarzi (2014), "The repair of environmentally relevant DNA double strand breaks caused by high linear energy transfer irradiation – No simple task.", DNA repair (Amst), 17:64-73. doi: 10.1016/j.dnarep.2014.01.014.

Mosconi, M., U. Giesen & F. Langner (2011), "53BP1 and MDC1 foci formation in HT-1080 cells for low- and high-LET microbeam irradiations.", Radiat. Envrion. Biophys. 50(3):345-352. doi:10.1007/s00411-011-0366-9.

Nagane, M. et al. (2021), “DNA damage response in vascular endothelial senescence: Implication for radiation-induced cardiovascular disease”, Journal of Radiation Research, Vol. 62/4, https://doi.org/10.1093/jrr/rrab032

Nenoi, M., B. Wang & G. Vares (2015), "In vivo radioadaptive response: A review of studies relevant to radiation-induced cancer risk.", Hum. Exp. Toxicol. 34(3):272-283. doi:10.1177/0960327114537537.

Nikitaki, Z. et al. (2016), "Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET).", Free Radiac. Res. 50(sup1):S64-S78, doi:10.1080/10715762.2016.1232484.

Nikjoo, H. et al. (2001), "Computational approach for determining the spectrum of DNA damage induced by ionizing radiation.", Radiat. Res. 156(5 Pt 2):577-83.

Nübel, T. et al. (2006), “Lovastatin protects human endothelial cells from killing by ionizing radiation without impairing induction and repair of DNA double-strand breaks”, Clinical Cancer Research, Vol. 12/3, American Association for Cancer Research, https://doi.org/10.1158/1078-0432.CCR-05-1903
Ojima, M., N. Ban, and M. Kai (2008), "DNA double-strand breaks induced by very low X-ray doses are largely due to bystander effects", Radiation Research, Vol. 170/3, Radiation Research Society, United States, https://doi.org/10.1667/RR1255.1

Okayasu, R. (2012a), "Repair of DNA damage induced by accelerated heavy ions-A mini review.", Int. J. Cancer. 130(5):991-1000. doi:10.1002/ijc.26445. Okayasu, R. (2012b), "Heavy ions — a mini review.", 1000:991-1000. doi:10.1002/ijc.26445.

Park, J. W. et al. (2022), "Metformin alleviates ionizing radiation-induced senescence by restoring BARD1-mediated DNA repair in human aortic endothelial cells", Experimental Gerontology, Vol. 160, Elsevier, Amsterdam, https://doi.org/10.1016/j.exger.2022.111706

Parris, C.N. et al. (2015), "Enhanced γ-H2AX DNA damage foci detection using multMagnification and extended depth of field in imaging flow cytometry.", Cytom. Part A. 87(8):717-723. doi:10.1002/cyto.a.22697.

Radulescu I., K. Elmroth & B. Stenerlöw (2006), "Chromatin Organization Contributes to Non-randomly Distributed Double-Strand Breaks after Exposure to High-LET Radiation.", Radiat. Res. 161(1):1-8. doi:10.1667/rr3094.

Rastogi, R. P. et al. (2010), "Molecular mechanisms of ultraviolet radiation-induced DNA damage and repair", Journal of nucleic acids, Hindawi Ltd, United States. https://doi.org/10.4061/2010/592980

Reddy, V. N. et al. (1998), "The effect of aqueous humor ascorbate on ultraviolet-B-induced DNA damage in lens epithelium", Investigative ophthalmology & visual science, Vol. 39/2, Arvo, pp. 344-350

Rogakou, E.P. et al. (1999), "Megabase Chromatin Domains Involved in DNA Double-Strand Breaks In Vivo.", J. Cell Biol, 146(5):905-16. doi: 10.1083/jcb.146.5.905.

Rogers, C. S. et al. (2004), "The effects of sub-solar levels of UV-A and UV-B on rabbit corneal and lens epithelial cells", Experimental eye research, Vol. 78/5, Elsevier Ltd, London, https://doi.org/10.1016/j.exer.2003.12.011

Rombouts, C. et al. (2013), "Differential response to acute low dose radiation in primary and immortalized endothelial cells", International Journal of Radiation Biology, Vol. 89/10, Informa, London, https://doi.org/10.3109/09553002.2013.806831

Rothkamm, K. et al. (2015), "Review DNA Damage Foci: Meaning and Significance.", Environ. Mol. Mutagen., 56(6):491-504, doi: 10.1002/em.21944.

Rothkamm, K. & M. Lo (2003), "Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses.", PNAS, 100(9):5057-62. doi:10.1073/pnas.0830918100.

Rübe, C.E. et al. (2008), "Cancer Therapy: Preclinical DNA Double-Strand Break Repair of Blood Lymphocytes and Normal Tissues Analysed in a Preclinical Mouse Model: Implications for Radiosensitivity Testing.", Clin. Cancer Res., 14(20):6546-6556. doi:10.1158/1078-0432.CCR-07-5147.

Russo, A. et al. (2015), "Review Article Genomic Instability: Crossing Pathways at the Origin of Structural and Numerical Chromosome Changes.", Envrion. Mol. Mutagen. 56(7):563-580. doi:10.1002/em.
Rydberg B, Heilbronn L, Holley WR, Lobrich M, Zeitlin C et al. 2002. Spatial Distribution and Yield of DNA Double-Strand Breaks Induced by 3-7 MeV Helium Ions in Human Fibroblasts. Radiat Res. 158(1):32-42.

Sabirzhanov, et al. (2020), "Irradiation-Induced Upregulation of miR-711 Inhibits DNA Repair and Promotes Neurodegeneration Pathways.", Int J Mol Sci. 21(15):5239. doi: 10.3390/ijms21155239.

Sage, E. & N. Shikazono (2017), "Free Radical Biology and Medicine Radiation-induced clustered DNA lesions: Repair and mutagenesis.", Free Radic. Biol. Med. 107(December 2016):125-135. doi:10.1016/j.freeradbiomed.2016.12.008.

Sasaki, H. et al. (1998), "TEMPOL protects against lens DNA strand breaks and cataract in the x-rayed rabbit", Investigative ophthalmology & visual sciences, Vol. 39/3, Arvo, Rockville, pp. 544-552

Schmal, Z. et al. (2019), "DNA damage accumulation during fractionated low-dose radiation compromises hippocampal neurogenesis", Radiotherapy and Oncology. 137:45-54. doi:10.1016/j.radonc.2019.04.021.

Sears, C. R. and J. J. Turchi (2012), "Complex cisplatin-double strand break (DSB) lesions directly impair cellular non-homologous end-joining (NHEJ) independent of downstream damage response (DDR) pathways", Journal of biological chemistry, Vol 287/29, The American Society for Biochemistry and Molecular Biology, Inc, USA, https://doi.org/10.1074/jbc.M112.344911

Shah, D.J., R.K. Sachs & D.J. Wilson (2012), "Radiation-induced cancer: A modern view." Br. J. Radiol. 85(1020):1166-1173. doi:10.1259/bjr/25026140.

Shelke, S. & B. Das (2015), "Dose response and adaptive response of non-homologous end joining repair genes and proteins in resting human peripheral blood mononuclear cells exposed to γ radiation.", (December 2014):365-379. doi:10.1093/mutage/geu081.

Sidjanin, D., S. Zigman and J. Reddan (1993), "DNA damage and repair in rabbitlens epithelial cells following UVA radiation", Current eye research, Vol. 12/9, Informa UK Ltd, Oxford, https://doi.org/10.3109/02713689309020382

Smith, J. et al. (2003), "Impact of DNA ligase IV on the delity of end joining in human cells.", Nucleic Acids Research. 31(8):2157-2167. doi:10.1093/nar/gkg317.

Smith, T.A. et al. (2017), "Radioprotective agents to prevent cellular damage due to ionizing radiation." Journal of Translational Medicine.15(1).doi:10.1186/s12967-017-1338-x.

Stewart, F. A. et al. (2012), "ICRP publication 118: ICRP statement on tissue reactions and early and late effects of radiation in normal tissues and organs – threshold doses for tissue reactions in a radiation protection context", Annals of the ICRP, Vol, 41/1-2, Elsevier Ltd, London, https://doi.org/10.1016/j.icrp.2012.02.001

Sudprasert, W., P. Navasumrit & M. Ruchirawat (2006), "Effects of low-dose γ radiation on DNA damage, chromosomal aberration and expression of repair genes in human blood cells.", Int. J. Hyg. Envrion. Health, 209:503-511. doi:10.1016/j.ijheh.2006.06.004.

Sutherland, B.M. et al. (2000), "Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation.", J. of Rad. Res. 43 Suppl(S):S149-52. doi: 10.1269/jrr.43.S149
Sylvester, C. B. et al. (2018), "Radiation-Induced Cardiovascular Disease: Mechanisms and Importance of Linear Energy Transfer", *Frontiers in cardiovascular medicine*, Vol. 5, Frontiers, https://doi.org/10.3389/fcvm.2018.00005

Terato, H. & H. Ide (2005), "Clustered DNA damage induced by heavy ion particles.", *Biol. Sci. Sp. 18*(4):206-215. doi:10.2187/bss.18.206.

Ungvari, Z. et al. (2013), "Ionizing radiation promotes the acquisition of a senescence-associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: role of increased DNA damage and decreased DNA repair capacity in microvascular radiosensitivity", The journals of gerontology. Series A, Biological sciences and medical sciences, Vol. 68/12, Oxford University Press, Oxford, https://doi.org/10.1093/gerona/glt057

Valentin, J.D.J (1998), "Chapter 1. Ann ICRP.", 28(4):5-7. doi:10.1016/S0146-6453(00)00002-6. http://www.ncbi.nlm.nih.gov/pubmed/10882804.

Venkatesh, P. et al. (2016), "Effect of chromatin structure on the extent and distribution of DNA double strand breaks produced by ionizing radiation; comparative study of hESC and differentiated cells lines.", *Int J. Mol. Sci.* 17(1). doi:10.3390/ijms17010058.

Wang, et al. (2021), "Ionizing Radiation-Induced Brain Cell Aging and the Potential Underlying Molecular Mechanisms.", *Cells*. 10(12):3570. doi: 10.3390/cells10123570

Ward, J. F. (1988), "DNA Damage Produced by Ionizing Radiation in Mammalian Cells: Identities, Mechanisms of Formation, and Reparability.", *Prog. Nucleic Acid Res. Mol. Biol.* 35(C):95-125. doi:10.1016/S0079-6603(08)60611-X.

Wolf, N. et al. (2008), "Radiation cataracts: Mechanisms involved in their long delayed occurrence but then rapid progression", *Molecular vision*, Vol. 14/34-35, Molecular Vision, Atlanta, pp. 274-285

Wu, L.J. et al. (1999), "Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian cells.", *Proc. Natl. Acad. Sci.* 96(9):4959-4964. doi:10.1073/pnas.96.9.4959.

Yamaguchi, H. et al. (2005), "Estimation of Yields of OH Radicals in Water Irradiated by Ionizing Radiation.", *J. of Rad. Res.* 46(3):333-41. doi: 10.1269/jrr.46.333.

Zhang, L. et al. (2017), "The inhibitory effect of minocycline on radiation-induced neuronal apoptosis via AMPKα1 signaling-mediated autophagy.", *Sci Rep*.7(1):16373. doi: 10.1038/s41598-017-16693-8
Relationship: 2837 – Energy Deposition leads to Abnormal Neural Remodeling

AOPs Referencing Relationship

<table><thead><tr><td>AOP Name</td><td>Adjacency</td><td>Weight of Evidence</td><td>Quantitative Understanding</td></tr></thead><tbody><tr><td>AOP: 483 - Deposition of Energy Leading to Learning and Memory Impairment</td><td>non-adjacent</td><td>Moderate</td><td>Low</td></tr></tbody></table>

Evidence Supporting Applicability of this Relationship

Most evidence is derived from *in vivo* studies, predominantly using rodent models, whereas evidence from dog models is low. The relationship is applicable in both sexes; however, adult males are used more often in animal studies. Limited studies demonstrate the relationship in preadolescent animals.

Taxonomic Applicability

<table><thead><tr><td>Term</td><td>Scientific Term</td><td>Evidence</td><td>Link</td></tr></thead><tbody><tr><td>mouse</td><td>Mus musculus</td><td>High</td><td>NCBI</td></tr><tr><td>rat</td><td>Rattus norvegicus</td><td>Low</td><td>NCBI</td></tr><tr><td>dog</td><td>Canis lupus familiaris</td><td>Low</td><td>NCBI</td></tr></tbody></table>

Life Stage Applicability

<table><thead><tr><td>Term</td><td>Evidence</td></tr></thead><tbody><tr><td>Adult</td><td>Moderate</td></tr><tr><td>Not Otherwise Specified</td><td>Low</td></tr><tr><td>Juvenile</td><td>Low</td></tr></tbody></table>

Sex Applicability

<table><thead><tr><td>Sex</td><td>Evidence</td></tr></thead><tbody><tr><td>Male</td><td>High</td></tr><tr><td>Female</td><td>Low</td></tr><tr><td>Unspecific</td><td>Low</td></tr></tbody></table>

Key Event Relationship Description

Energy deposition through ionizing radiation can lead to chemical changes including bond breakages and the generation of by-products, such as free radicals from water hydrolysis, which can change cellular homeostasis (Einor et al., 2016; Martinez-López & Hande, 2020; Reisz et al., 2014). This energy can come in many forms (i.e., gamma rays, X-rays, alpha particles, heavy ions, protons), to produce a range in complexity of damage (Drobny, 2013). When deposited onto neurons, oxidative stress can affect neuronal signaling through the induction of alterations to the neuronal architecture and synaptic activity. The energy can further cause necrosis and demyelination and decrease neurogenesis and synaptic complexity; these together are important to maintain the integrity of the neurons (Cekanaviciute et al., 2018; J. R. Fike et al., 1984; Hladik & Tapio, 2016). Furthermore, there can also be disruptions in neuronal signaling, as well as changes to drebrin cluster and postsynaptic density proteins (PSD), which are known to regulate dendritic spine morphogenesis. Together these can lead to abnormal neural remodeling (Takahashi et al., 2003).
The strategy for collating the evidence to support the relationship is described in Kozbenko et al 2022. Briefly, a scoping review methodology was used to prioritize studies based on a population, exposure, outcome, endpoint statement.

Overall Weight of Evidence: Moderate

**Biological Plausibility**

Multiple reviews provide support of biological plausibility between deposition of energy and neuron remodeling. Numerous studies examine the effects of multiple radiation sources including gamma rays, X-rays, protons, heavy ions, alpha particles, and neutrons, on both in vivo and in vitro model systems. The collected data demonstrate changes in the physical and electrophysiological properties of neurons in response to ionizing radiation exposure (Cekanaviciute et al., 2018; Hladik & Tapio, 2016). Irradiation of the brain induces oxidative stress and inflammation, which is linked to the occurrence of neuronal alterations within the hippocampus, an important structure in the process of learning and memory (Jarrard, 1993; Monje et al., 2003; Rola et al., 2007; Lalkovičová et al., 2022). There is no clear understanding on how deposited energy directly affects neuron integrity; however, immature neurons are found to be more radiosensitive and exhibit significant changes in spine density, dendritic spine length, protein clustering, and decreased cell proliferation, leading to reduced dendritic complexity (Manda et al., 2008a; Mizumatsu et al., 2003; Okamoto et al., 2009; Rola et al., 2005; Shirai et al., 2013). Many neurodegenerative conditions are related to changes in synaptic plasticity, including changes in neuronal connectivity, action potential, and synaptic protein levels (Vipan Kumar Parihar & Limoli, 2013). These alterations occur primarily within the hippocampus and dentate gyrus, two regions of the mammalian brain where adult neurogenesis can occur (Ming & Song, 2011) following ionizing radiation exposure.

**Empirical Evidence**

The empirical evidence collected for this KER stems from research using both in vitro and in vivo models. Abnormal neural remodeling encompasses changes to the physical and/or electrophysiological properties of neurons (Acharya et al., 2019; Klein et al., 2021). Most of the evidence examines the effects of moderate (0.1-0.5 Gy) or high (>2 Gy) dose irradiation on both in vivo and in vitro rodent models. Studies using X-ray and/or heavy ion irradiation ranging from 0.05 – 30 Gy identify similar alterations in dendritic spine structure, neurogenesis, and apoptosis (Allen et al., 2015; Cekanaviciute et al., 2018; J. R. Fike et al., 1984; Hladik & Tapio, 2016; Kiffer et al., 2020; Manda et al., 2008a; Mizumatsu et al., 2003; Okamoto et al., 2009; Vipan K. Parihar et al., 2016; Vipan K. Parihar; Rola et al., 2005; Tiller-Borcich et al., 1987; Panagiotakos et al., 2007). The low dose evidence (<0.1 Gy) stems from studies from heavy ion or alpha particle irradiation of hippocampal mouse tissue (Krukowski et al., 2018b; Vipan K. Parihar et al., 2016; Vipan K. Parihar, Allen, et al., 2015). High dose studies (2 Gy up to 90 Gy) show more extreme changes in neuron integrity through decreased CNPase levels, increased necrosis and reduced cytoskeleton proteins (Chiang et al., 1993; Jiang et al., 2014; Shirai et al., 2013).

**Dose Concordance**

Several studies support a dose-concordant relationship between energy deposition and abnormal neural remodeling. Dose-dependent increases in neuronal apoptosis/necrosis and decreases in F-actin, drebrin and synapsin I protein clusters are observed in a range of doses from 0.5 Gy to 10 Gy (Mizumatsu et al., 2003; Okamoto et al., 2009). Studies that examine changes in dendritic spines exhibit dose-dependent decreases in filopodia spines at 0.1 Gy to 10 Gy (Vipan K. Parihar, Pasha, et al., 2015; Vipan Kumar Parihar & Limoli, 2013). High-dose irradiation resulted in more severe damage in the white matter, including edema, demyelination, and necrosis at 15 Gy (Tiller-Borcich et al., 1987), as well as significant decreases in protein clusters at doses greater than 30 Gy (Shirai et al., 2013).
Across different types of radiation stressors, the data exhibits consistent dose-dependent changes to neuron integrity but of different magnitudes. A study examining the effects of 56Fe and 12C ions observed dose-dependent decreases in neurogenesis within each age group and type of irradiation. Overall, 56Fe irradiation of 9-month-old mice revealed a greater decrease in proliferating cells and immature neurons compared to 12C ions, except at 3 Gy, where there was a slightly greater number of proliferating cells (Rola et al., 2005). Similarly, compared to 16O particles, Parihar et al. found greater decreases in the numbers of dendritic branches and branch points when exposed to 48Ti particles; however, 16O particles led to a greater decrease in spine density (Parihar et al., 2015).

## Time Concordance

Multiple studies support time concordance between deposition of energy and abnormal neural remodeling. Most of these studies identify persistent, time-dependent deterioration of the neuronal structure up to 4 months post-irradiation at doses ranging from 0.05 – 30 Gy and using radiation types of gamma rays, protons, X-rays, and heavy ions (Chakraborti et al., 2012; Vipan K. Parihar et al., 2016; Vipan K. Parihar, Allen, et al., 2015; Vipan K. Parihar, Pasha, et al., 2015; Vipan Kumar Parihar & Limoli, 2013). At 9 months post-irradiation (0.5 Gy of protons), Bellone et al. (2015) observed enhanced synaptic excitability, indicative of increases in synaptic density, and Rola et al., (2005) found that the number of proliferating neural precursor cells was lower than that at 3 months (1 and 3 Gy of 56Fe ions). At 24 h to 15 months post-irradiation (25 Gy of X-rays), Panagiotakos et al. (2007) showed unrepairable damage to both the subventricular zone and neural stem cell compartment in the female rat model. A study at 30 months (15 Gy of X-rays) revealed a reduction in computed tomography (CT) density, indicative of edema, demyelination, axonal swelling, and/or necrosis (J. R. Fike et al., 1984). In examining different radiation types, the time-response relationship could not be clearly identified due to differences in experimental design across studies. However, two studies analyzing PSD-95 expression, a protein involved in regulating synaptic plasticity, at 10- and 30-days post-irradiation found that 1 Gy of gamma ray irradiation led to similar time- dependent increases in PSD-95 compared to 1 Gy of proton irradiation (Vipan K. Parihar, Pasha, et al., 2015; Vipan Kumar Parihar & Limoli, 2013).

## Incidence Concordance

No available data.

## Essentiality

As deposition of energy is a physical stressor, it cannot be blocked by chemicals, however, it can be shielded (Al Zaman & Nizam, 2022). Further research is required to determine the effects of shielding radiation on abnormal neural remodeling. Since deposited energy initiates events immediately, the removal of deposited energy, a physical stressor, also supports the essentiality of the key event. Studies that do not deposit energy are observed to have no downstream effects.

## Uncertainties and Inconsistencies

* One study shows that at a high dose of 90 Gy (X-rays), hippocampal dendritic spine length is increased compared to the control (Shirai et al., 2013). Kiffer et al. found a decrease in spine length in the CA1 subregion of the hippocampus; however, there was an increase in spine length and dendritic complexity in the dorsal dentate gyrus after exposure to 0.5 Gy 1H and 0.1 Gy 16O (Kiffer et al., 2020). Further research involving different regions of the brain is required to identify the effects of deposition of energy on the dentate gyrus.

* The study by Krukowski et al. (2018a), as highlighted in a review by Cekanaviciute et al. (2018), found a lack of cellular changes in hippocampal synapse loss and microgliosis in females subjected to low dose ionizing radiation. Another study highlighted in this review showed greater reduction in new neuronal survival in male than female mice in response to 28Si irradiation (Whoolery et al., 2017). Additional data is required to determine if these differences are sex-related or due to other factors. There is a lack of studies performed on female subjects to identify specific sex-related effects of deposited energy on neuron integrity.
* Previous studies have found transient changes in neurogenesis after exposure to 56Fe ions at varying doses ranging from 10 cGy to 1 Gy (DeCarolis et al., 2014; Miry et al., 2021). These studies found early decreases in neurogenesis, although DeCarolis et al. reported that this reduction returned to normal as early as 7 days post-irradiation. In a separate study, Miry et al. (2021) found that at 12 months post-exposure, neurogenesis levels significantly exceeded controls. Other inconsistent studies include Acharya et al. (2019) and Bellone et al. (2015); the former reported decreases in CA1 pyramidal neuron excitability after exposure to 18 cGy of neutron radiation, whereas the latter study reported increases in post-synaptic excitability within CA1 neurons after 0.5 Gy of proton irradiation.

* Chien et al. (2015) found no changes to survival or number of synapses in hippocampal neurons five days after 0.2 Gy of electron radiation.

## Known modulating factors

<table><thead><tr><td>Modulating Factor</td><td>Details</td><td>Effects on the KER</td><td>References</td></tr></thead><tbody><tr><td>Drug</td><td>SB415286 – a potent and selective cell-permeable, ATP-competitive GSK-3β inhibitor, as GSK-3β induces apoptosis in response to various conditions</td><td>Treatment with SB415286 provided significant neuroprotection against radiation necrosis within the brain at 45 Gy.</td><td>Jiang et al., 2014</td></tr><tr><td>Diet</td><td>N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) – a melatonin metabolite which has antioxidant properties.</td><td>AMFK treatment ameliorated levels of reactive oxygen species, and increased number of immature neurons and proliferating cells post-irradiation in vivo. Without the treatment, exposure to radiation led to a significant decrease in DCX positive cells by 81% and Ki-67 positive cells by 86%. (AMFK) treatment provided protection to immature neurons by 45.38% and proliferating cells by 52.35%.</td><td>Manda et al., 2008b</td></tr><tr><td>Drug</td><td>PLX5622-1200 ppm (PLX) diet that contains CSF1-R (colony stimulating factor 1 receptor) inhibitor that induces depletion of microglia within 3 days.</td><td>The PLX diet was able to significantly increase the levels of the presynaptic protein, synapsin 1 after exposure to helium irradiation.</td><td>Krukowski et al., 2018b</td></tr></tbody></table>

## Quantitative Understanding of the Linkage

The table below provides some representative examples of quantitative linkages between the two key events. It was difficult to identify a general trend across all the studies due to differences in experimental design and reporting of the data. All data is statistically significant unless otherwise stated.

### Dose Concordance

<table><thead><tr><td>Reference</td><td>Experimental Description</td><td>Results</td></tr></thead><tbody><tr><td>Acharya et al., 2019</td><td>In vivo. Male mice were irradiated with 252Cf neutrons at a dose rate of 1 mGy per day for 180 days. The mice were then sacrificed, and brains were removed to conduct whole-cell electrophysiology on the pyramidal neurons in the dorsal hippocampus.</td><td>18 cGy of neutron radiation caused pyramidal neurons to be more hyperpolarized with a mean difference score (Mdiff) of -2.56 mV. CA1 pyramidal neurons exhibited a decrease in excitability (Mdiff = 30.8 pA) at 18 cGy.</td></tr><tr><td>Parihar & Limoli, 2013</td><td>In vivo. Male transgenic mice were exposed to cranial gamma irradiation at a dose of 1 or 10 Gy. This was done using a 137Cs irradiator at a dose rate of 2.07 Gy/min. After tissue harvesting, fluorescent imaging was performed on hippocampal neurons expressing the enhanced green fluorescent protein (EGFP) transgene to analyze changes in dendritic tree.</td><td>Dose-dependent reductions in the number of dendritic branch points, length, and area at both 10 and 30 days after gamma irradiation. Significant increase in PSD-95 (postsynaptic scaffolding protein) expression of 2.7-fold at 10 Gy at both time-points, and 1.7- and 2.2-fold increases for 1 Gy at 10 and 30 days, respectively. 1.8- and 3.7-fold decrease in filopodia at 1 and 10 Gy.</td></tr><tr><td>Parihar, Pasha et al., 2015</td><td>In vivo. Male transgenic mice were exposed to whole body proton irradiation at a dose of 0.1 or 1 Gy. This was done using 250 MeV plateau phase protons at a dose rate of 0.25 Gy/min. Mice were sacrificed 10 or 30 days post-irradiation, then fluorescent imaging was performed on hippocampal neurons expressing the EGFP transgene to analyze changes in dendritic tree.</td><td>Dose-dependent increases in postsynaptic density protein 95 (PSD-95) expression of 1.8- (0.1 Gy) and 2.1-fold (1 Gy) at 30 days in the dentate gyrus after proton irradiation. Dose-dependent reduction in synaptophysin expression of 1.3- (0.1 Gy) and 1.9-fold (1 Gy) in the dentate hilus. Dose-dependent and time-dependent decreases in number of filopodia spines (2.9- and 3.7-fold decreases at 10 and 30 days at 1 Gy).</td></tr></tbody></table>
<table>
  <tr>
    <td></td>
    <td>Immunohistochemistry was also performed to measure synaptic proteins.</td>
    <td></td>
  </tr>
  <tr>
    <td>Mizumatsu et al., 2003</td>
    <td>In vivo. Male mice were exposed to cranial X-irradiation at 1, 2, 5 and 10 Gy (rate of 175 cGy/min). Confocal microscopy was then used to assess changes in neurogenesis and neuronal proliferation.</td>
    <td>Numbers of immature neurons in the subgranular zone reduced by 36, 51, 56, and 67% at 1, 2, 5, and 10 Gy, respectively, indicative of decreased neurogenesis. Decrease in number of neural cells by 41, 53, and 61% at 2, 5, and 10 Gy, indicative of increased apoptosis.</td>
  </tr>
  <tr>
    <td>Rola et al., 2005</td>
    <td>In vivo. Male mice received whole-body irradiation with iron or carbon ions at doses of 1, 2 or 3 Gy. These were delivered at dose rates of 0.87 ± 0.16 Gy/min and 1.23 ± 0.07 Gy/min for iron and carbon ions, respectively. Confocal microscopy and immunohistochemistry were then performed to analyze proliferating neurons and neurogenesis.</td>
    <td>56Fe irradiation led to 48% (1 Gy, 3 months old), 76% (3 Gy, 3 months), 81% (1 Gy, 9 months old), and 65% (3 Gy, 9 months) decreases in proliferating (Ki67-positive) cells in the dentate subgranular zone and 34% (1 Gy, 3 months), 71% (3 Gy, 3 months), 59% (1 Gy, 9 months), and 89% (3 Gy, 9 months) decreases in immature neurons (DCx-positive cells). 12C irradiation led to 25% (1 Gy, 9 months) and 72% (3 Gy, 9 months) decreases in Ki67-positive cells and 20% (1 Gy, 9 months) and 62% (3 Gy, 9 months) decreases in DCx-positive cells.</td>
  </tr>
  <tr>
    <td>Chiang et al., 1993</td>
    <td>In vivo. Male mice were exposed to 2, 8, 20, 30, 36 and 45 Gy of X-irradiation at a dose rate of 238 cGy/min. ELISA was performed to analyze CNPase levels.</td>
    <td>CNPase (myelin-associated enzyme) levels were decreased to approximately 85% after 20, 30, and 45 Gy before increasing to normal values at 15 days. At 60 days, 20 Gy CNPase levels peaked with an approximate increase to 110% before decreasing to 84% at 180 days. At 30 Gy, CNPase levels peaked after 120 days at 86% before decreasing to 64% after 270 days. At 45 Gy, CNPase levels continued to decrease to 73% 180 days post-irradiation.</td>
  </tr>
  <tr>
    <td>Allen et al., 2015</td>
    <td>In vivo. Male mice received whole-body iron irradiation at 0.5 Gy (dose rate was not indicated). Golgi staining was performed for spine analysis and Sholl analysis for dendritic morphology quantification.</td>
    <td>Dentate gyrus spine density 34% decreased after 0.5 Gy 56Fe irradiation. Dentate gyrus dendritic length decreased by 20% at 79.0 and 100.8 µm from soma and 27% at 120.9 and 130.2 µm from soma. CA1 basal spine density decreased by 32%. A peak decrease of 49% in CA1 dendritic length was observed at 77 µm from soma. CA3 apical spine density decreased by 20%.</td>
  </tr>
  <tr>
    <td>Vipan K. Pariar et al., 2016</td>
    <td>In vivo. Male transgenic mice and male rats were exposed to charged particles (16O and 48Ti at 600 MeV/n) at dose rates between 0.05 and 0.25 Gy/min. After tissue harvesting, confocal imaging was used for EGFP expressing neurons to assess neuronal morphometry 15 weeks post exposure.</td>
    <td>Total dendritic length was decreased by 25%, 28%, 33%, and 34%; number of dendritic branch points decreased by 25%, 27%, 25%, and 27% respectively; and number of dendritic branches by 15%, 83%, 32%, and 28%, for 16O – 5 cGy, 16O – 30 cGy, 48Ti – 5 cGy, and 48Ti – 30 cGy, respectively.</td>
  </tr>
  <tr>
    <td>Shirai et al., 2013</td>
    <td>In vitro. Hippocampal cultures were exposed to 30, 60 and 90 Gy of X-irradiation at a rate of 86.7 cGy/min. Green fluorescent protein (GFP) transfection was used to analyze the effects of X-ray irradiation on dendritic spine morphology and density. Fluorescent immunohistochemistry was used to evaluate cytoskeletal proteins within dendritic spines.</td>
    <td>Significant increase in dendritic spine length at 90 Gy by 1.2-fold. No change in width of dendritic spines. Number of F-actin clusters decreased by 1.4- (30 Gy), 1.57- (60 Gy), and 1.4-fold (90 Gy). Number of drebrin clusters decreased by 1.3- (30 and 60 Gy) and 1.4-fold (90 Gy).</td>
  </tr>
  <tr>
    <td>Okamoto et al., 2009</td>
    <td>In vitro. Primary neurons from the hippocampi of fetal rats were exposed to 0.5, 4 and 10 Gy of X-irradiation. Immunofluorescence was performed to analyze synaptic proteins and TUNEL staining was performed to identify neuronal apoptosis.</td>
    <td>Significant increase in apoptosis of neurons of 1.3- (0.5 Gy), 1.9- (4 Gy), and 2.5-fold (10 Gy) at 7 days post-irradiation and 1.4- (0.5 Gy), 2.3- (4 Gy), and 2.6-fold (10 Gy) at 14 days post-irradiation. Significant decrease in F-actin clusters of 1.4-fold (10 Gy) at 7 days, and 1.4- (4 Gy) and 1.5-fold (10 Gy) at 14 days. Significant decrease in drebrin of 1.8-fold (10 Gy) at 7 days, and 1.7- (4 Gy) and 1.5-fold (10 Gy) at 14 days. Significant decrease in Synapsin-I of 1.1-fold (10 Gy) at 14 days.</td>
  </tr>
  <tr>
    <td>Vipan K. Pariar, Allen, et al., 2015</td>
    <td>In vivo. Male transgenic mice (with EGFP transgene) were exposed to 5-30 cGy of charged particles (16O and 48Ti) at dose rates between 0.5 and 1.0 Gy/min. After tissue harvesting, confocal microscopy, imaging and neuronal morphometry were performed to evaluate dendritic complexity and spine density 8 weeks post radiation exposure.</td>
    <td>Significant reductions in number of dendritic branches by 1.5- (16O – 30 cGy), 1.8- (48Ti – 5 cGy), and 1.7-fold (48Ti – 30 cGy), number of branch points by 1.5- (16O – 30 cGy), 1.4- (48Ti – 5 cGy), and 1.8-fold (48Ti – 30 cGy), total dendritic length by 1.5- (16O – 5 cGy), 1.8- (16O – 30 cGy), 1.5- (48Ti – 5 cGy), and 1.7- fold (48Ti – 30 cGy), number of spines by 1.5- (16O – 5 cGy), 1.8- (16O – 30 cGy), 1.7- (48Ti – 5 cGy), 1.9-fold (48Ti – 30 cGy), and spine density by 1.7- (16O – 5 cGy), 1.6- (16O – 30 cGy), 1 and 1.5-fold (48Ti – 5 and 30 cGy). Significant increase in PSD-95 puncta by 1.5-fold (all doses and radiation types) 8 weeks post-irradiation.</td>
  </tr>
  <tr>
    <td>Kiffer et al., 2020</td>
    <td>In vivo. Male mice received whole-body 1H and 16O irradiation at 0.5 and 0.1 Gy, respectively. The dose rates were 18-19 cGy/min (1H irradiation) and 18-33 cGy/min (16O irradiation). Golgi staining was performed</td>
    <td>Dentate gyrus dendritic complexity increased by 1.4-fold after 0.5 Gy (1H) and 0.1 Gy (16O) combined irradiation.</td>
  </tr>
</table>
<table><tr><td></td><td>for dendritic morphology quantification and spine analyses.</td><td></td></tr><tr><td>Klein et al., 2021</td><td>In vivo. Male mice received whole-body simulated galactic cosmic irradiation (1H, 16O, 4He, 28Si, 56Fe) at 5 and 30 cGy. The dose rate was 5 cGy/min for the mixed-ion simulated galactic cosmic radiation (GCR). Electrophysiological measurements were then taken to assess changes in synaptic signaling within the CA1 region of the hippocampus.</td><td>30 cGy of mixed-ion GCR causes CA1 pyramidal neurons to have elevated amplitudes in the spontaneous inhibitory postsynaptic currents (sIPSC) with a mean difference score (Mdiff) of 5.54 pA. There was also a decrease in the sIPSC rise times (Mdiff = -0.45 ms). These results show that GCR leads to enhanced inhibitory synaptic signaling.</td></tr></table>

Time Concordance

<table><thead><tr><td>References</td><td>Experimental Description</td><td>Results</td></tr></thead><tbody><tr><td>Vipan K. Parihar, Pasha, et al., 2015</td><td>In vivo. Male transgenic mice were exposed to whole body proton irradiation at a dose of 0.1 or 1 Gy. This was done using 250 MeV plateau phase protons at a dose rate of 0.25 Gy/min. Mice were sacrificed at 10 or 30 days post-irradiation, then fluorescent imaging was performed on hippocampal neurons expressing the EGFP transgene to analyze changes in dendritic tree. Immunohistochemistry was also performed to measure synaptic proteins.</td><td>Time-dependent increase in PSD-95 of 1.6- (10 days) and 2.1-fold (30 days). Time-dependent decrease in synaptophysin of 1.4- (10 days) and 1.9-fold (30 days) in the dentate hilus.</td></tr><tr><td>Bellone et al., 2015</td><td>In vivo. Male mice received whole-body irradiation with 0.5 Gy of 150 MeV protons at a rate of 1.5-2.5 Gy/min. Electrophysiological experiments were conducted to measure synaptic response changes.</td><td>After 9 months, exposure to 0.5 Gy of proton irradiation resulted in an increase in postsynaptic excitability by 53% at 1.50 mA stimulation. Synaptic efficacy was increased by 118%. fEPSP (excitatory postsynaptic potential) slopes increased approximately 15 – 46% at least 60 min post-stimulation. There were no changes in presynaptic ability.</td></tr><tr><td>J. R. Fike et al., 1984</td><td>In vivo. Male beagle dogs were exposed to 10, 15, 30 Gy of X-irradiation at a rate of 3 Gy/min. Computed tomography (CT) scan and histological examination were performed to identify differences in tissue density within the cerebrum.</td><td>Animals receiving 10 Gy had no response up to 12 months. Time to maximum computed tomography (CT) response was 6.3 months for 15 Gy and 5.2 months for 30 Gy.</td></tr><tr><td>Chakraborti et al., 2012</td><td>In vivo. Male mice were irradiated with a single dose of 10 Gy from a 137Cs source at a rate of 1.67 Gy/min. After harvesting, neurons underwent Golgi staining to analyze changes in spine density and morphology.</td><td>10 Gy of gamma irradiation led to a time-dependent decrease in spine density in the dentate gyrus by 11.9% (1 week) and 26.9% (1 month), a significant reduction in proportion of mushroom spines by 24.3% (1 week) and 15.7% (1 month), a significant decrease in the number of thin spines by 11.49% (1 week) and 13.2% (1 month), and a significant increase in number of mushroom spines by 12.1% (1 week) and 25.3% (1 month).</td></tr><tr><td>Okamoto et al., 2009</td><td>In vitro. Primary neurons from the hippocampi of fetal rats were exposed to 0.5, 4 and 10 Gy of X-rays. Immunofluorescence was performed to analyze synaptic proteins and TUNEL staining was performed to identify neuronal apoptosis.</td><td>Significant increase in apoptosis of neurons of 1.3- (0.5 Gy), 1.9- (4 Gy), and 2.5-fold (10 Gy) at 7 days post-irradiation and 1.4- (0.5 Gy), 2.3- (4 Gy), and 2.6-fold (10 Gy) at 14 days post-irradiation. Significant decrease in F-actin clusters of 1.4-fold (10 Gy) at 7 days, and 1.4- (4 Gy) and 1.5-fold (10 Gy) at 14 days. Significant decrease in drebrin of 1.8-fold (10 Gy) at 7 days, and 1.7- (4 Gy) and 1.5-fold (10 Gy) at 14 days. Significant decrease in Synapsin-I of 1.1-fold (10 Gy) at 14 days.</td></tr><tr><td>Panagiotakos et al., 2007</td><td>In vivo. Female Sprague Dawley rat brains were irradiated with X-rays at 25 Gy (117.5 cGy/min) while shielding the olfactory bulb. Immunohistochemistry, stereological analysis, fluorescence intensity quantification, electron microscopy and magnetic resonance imaging were performed to assess long term radiation damage 24 h to 15 months post irradiation.</td><td>The number of BrdU+ cells in the subventricular zone (SVZ) decreased significantly 15 months post irradiation (average number of BrdU+cells was 5,541+/-624 compared to the control group of 34,680+/-9,413). Doublecortin (DCX)-expressing neuroblasts were lost permanently immediately post irradiation.</td></tr></tbody></table>
References

Acharya, M. M. et al. (2019), "New concerns for neurocognitive function during deep space exposures to chronic, low dose-rate, neutron radiation", eNeuro, Vol. 6/4, Society for Neuroscience, Washington, https://doi.org/10.1523/ENEURO.0094-19.2019.

Allen, A. R. et al. (2015), "56Fe Irradiation Alters Spine Density and Dendritic Complexity in the Mouse Hippocampus", Radiation Research, Vol. 184/6, BioOne, Washington, https://doi.org/10.1667/RR14103.1.

Al Zaman, M. A. and Q. M. R. Nizam. (2022), "Study on Shielding Effectiveness of a Combined Radiation Shield for Manned Long Termed Interplanetary Expeditions", Journal of Space Safety Engineering, Vol. 9/1, Elsevier, Amsterdam, https://doi.org/10.1016/j.jsse.2021.12.003.

Bellone, J. A. et al. (2015), "A single low dose of proton radiation induces long-term behavioral and electrophysiological changes in mice", Radiation Research, Vol. 184/2, BioOne, Washington, https://doi.org/10.1667/RR13903.1.

Cekanaviciute, E., S. Rosi and S. V. Costes. (2018), "Central Nervous System Responses to Simulated Galactic Cosmic Rays", International Journal of Molecular Sciences, Vol. 19/11, Multidisciplinary Digital Publishing Institute (MDPI), Basel, https://doi.org/10.3390/IJMS19113669.

Chakraborti, A. et al. (2012), "Cranial Irradiation Alters Dendritic Spine Density and Morphology in the Hippocampus", PLOS ONE, Vol. 7/7, Public Library of Science, San Francisco, https://doi.org/10.1371/JOURNAL.PONE.0040844.

Chiang, C. S., W. H. McBride and H. Rodney Withers. (1993), "Myelin-associated changes in mouse brain following irradiation", Radiotherapy and Oncology, Vol. 27/3, Elsevier, Amsterdam, https://doi.org/10.1016/0167-8140(93)90079-N.

DeCarolis, N. A. et al. (2014), "56Fe particle exposure results in a long-lasting increase in a cellular index of genomic instability and transiently suppresses adult hippocampal neurogenesis in vivo", Life Sciences in Space Research, Vol. 2, https://doi.org/10.1016/j.lssr.2014.06.004.

Dhikav, V. and K. Anand. (2012), "Hippocampus in health and disease: An overview", Annals of Indian Academy of Neurology, Vol. 15/4, Wolters Kluwer, Alphen aan den Rijn, https://doi.org/10.4103/0972-2327.104323.

Drobny, J. G. (2013), "Introduction", Ionizing Radiation and Polymers (pp. 1–10), Elsevier, Amsterdam, https://doi.org/10.1016/B978-1-4557-7881-2.00001-8.

Einor, D. et al. (2016), "Ionizing radiation, antioxidant response and oxidative damage: A meta-analysis", Science of The Total Environment, Vols. 548–549, Elsevier, Amsterdam, https://doi.org/10.1016/j.scitotenv.2016.01.027.

Fike, J. R. et al. (1984), "Computed Tomography Analysis of the Canine Brain: Effects of Hemibrain X Irradiation", Radiation Research, Vol. 99/2, Allen Press, Lawrence, https://doi.org/10.2307/3576373.

Hainmueller, T. and M. Bartos. (2020), "Dentate gyrus circuits for encoding, retrieval and discrimination of episodic memories", Nature Reviews Neuroscience, Vol. 21/3, Springer Nature, Berlin, https://doi.org/10.1038/s41583-019-0260-z.

Harris, K. M. and J. K. Stevens. (1989), "Dendritic spines of CA 1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics", Journal of
Hladik, D. and S. Tapio. (2016), "Effects of ionizing radiation on the mammalian brain", Mutation Research - Reviews in Mutation Research, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.08.003.

HR, C. C. M. W. W. (1993), "Radiation-induced astrocytic and microglial response in mouse brain", papers3://publication/uuid/729C572E-9A43-4EF4-A184-8A9DAD8CBC38.

Jarrard, L. E. (1993), "On the role of the hippocampus in learning and memory in the rat", Behavioral and Neural Biology, Vol. 60/1, Academic Press, Cambridge, https://doi.org/10.1016/0163-1047(93)90664-4.

Jiang, X. et al. (2014), "A GSK-3β Inhibitor Protects Against Radiation Necrosis in Mouse Brain", International Journal of Radiation Oncology*Biology*Physics, Vol. 89/4, Elsevier, Amsterdam, https://doi.org/10.1016/J.IJROBP.2014.04.018.

Kiffer, F. et al. (2020), "Late Effects of 1H + 16O on Short-Term and Object Memory, Hippocampal Dendritic Morphology and Mutagenesis", Frontiers in Behavioral Neuroscience, Vol. 14, Frontiers Media S.A., https://doi.org/10.3389/fnbeh.2020.00096.

Klein, P. M. et al. (2021), "Detrimental impacts of mixed-ion radiation on nervous system function", Neurobiology of Disease, Vol. 151, Elsevier, https://doi.org/10.1016/j.nbd.2021.105252.

Krukowski, K. et al. (2018a), "Female mice are protected from space radiation-induced maladaptive responses", Brain, Behavior, and Immunity, Vol. 74, Academic Press Inc., https://doi.org/10.1016/j.bbi.2018.08.008.

Krukowski, K. et al. (2018b), "Temporary microglia-depletion after cosmic radiation modifies phagocytic activity and prevents cognitive deficits", Scientific Reports 2018 8:1, Vol. 8/1, Nature Publishing Group, Berlin, https://doi.org/10.1038/s41598-018-26039-7.

Lalkovičová, M. (2022), "Neuroprotective agents effective against radiation damage of central nervous system", Neural Regeneration Research, Vol. 17/9, https://doi.org/10.4103/1673-5374.335137.

Manda, K., M. Ueno and K. Anzai. (2008a), "Memory impairment, oxidative damage and apoptosis induced by space radiation: Ameliorative potential of α-lipoic acid", Behavioural Brain Research, Vol. 187/2, Elsevier, Amsterdam, https://doi.org/10.1016/j.bbr.2007.09.033.

Manda, K., M. Ueno and K. Anzai. (2008b), "Space radiation-induced inhibition of neurogenesis in the hippocampal dentate gyrus and memory impairment in mice: ameliorative potential of the melatonin metabolite, AFMK", Journal of Pineal Research, Vol. 45/4, John Wiley & Sons, Inc., Hoboken, https://doi.org/10.1111/j.1600-079X.2008.00611.x.

Martinez-López, W. and M. P. Hande. (2020), "Health effects of exposure to ionizing radiation", Advanced Security and Safeguarding in the Nuclear Power Industry (pp. 81–97), Elsevier, Amsterdam, https://doi.org/10.1016/B978-0-12-818256-7.00004-0.

Ming, G. and H. Song. (2011), "Adult Neurogenesis in the Mammalian Brain: Significant Answers and Significant Questions", Neuron, Vol. 70/4, NIH Public Access, https://doi.org/10.1016/J.NEURON.2011.05.001.
Miry, O. et al. (2021), "Life-long brain compensatory responses to galactic cosmic radiation exposure", Scientific Reports, Vol. 11/1, https://doi.org/10.1038/s41598-021-83447-y.

Mizumatsu, S. et al. (2003), "Extreme sensitivity of adult neurogenesis to low doses of X-irradiation", Cancer Research, Vol. 63/14.

Monje, M. L., H. Toda and T. D. Palmer. (2003), "Inflammatory Blockade Restores Adult Hippocampal Neurogenesis", Science, Vol. 302/5651, American Association for the Advancement of Science, Washington, https://doi.org/10.1126/SCIENCE.1088417.

Okamoto, M. et al. (2009), "Effect of radiation on the development of immature hippocampal neurons in vitro", Radiation Research, Vol. 172/6, BioOne, Washington, https://doi.org/10.1667/RR1741.1.

Panagiotakos, G. et al. (2007), "Long-Term Impact of Radiation on the Stem Cell and Oligodendrocyte Precursors in the Brain", PLoS ONE, Vol. 2/7, https://doi.org/10.1371/journal.pone.0000588.

Parihar, V. K. et al. (2016), "Cosmic radiation exposure and persistent cognitive dysfunction", Scientific Reports, Vol. 6/June, Nature Publishing Group, Berlin, https://doi.org/10.1038/srep34774.

Parihar, V. K. et al. (2015), "What happens to your brain on the way to Mars", Science Advances, Vol. 1/4, American Association for the Advancement of Science, Washington, https://doi.org/10.1126/SCIADV.1400256.

Parihar, V. K. et al. (2014), "Persistent changes in neuronal structure and synaptic plasticity caused by proton irradiation", Brain Structure and Function 2014 220:2, Vol. 220/2, Springer Nature, Berlin, https://doi.org/10.1007/S00429-014-0709-9.

Parihar, V. K. and C. L. Limoli. (2013), "Cranial irradiation compromises neuronal architecture in the hippocampus", Proceedings of the National Academy of Sciences of the United States of America, Vol. 110/31, National Academy of Sciences, Washington, https://doi.org/10.1073/pnas.1307301110.

Reisz, J. A. et al. (2014), "Effects of Ionizing Radiation on Biological Molecules—Mechanisms of Damage and Emerging Methods of Detection", Antioxidants & Redox Signaling, Vol. 21/2, Mary Ann Liebert, Inc., New Rochelle, https://doi.org/10.1089/ars.2013.5489.

Rola, R. et al. (2005), "High-LET radiation induces inflammation and persistent changes in markers of hippocampal neurogenesis", Radiation Research (Volume 164, pp. 556–560), BioOne, Washington, https://doi.org/10.1667/RR3412.1.

Rola, R. et al. (2007), "Lack of extracellular superoxide dismutase (EC-SOD) in the microenvironment impacts radiation-induced changes in neurogenesis", Free Radical Biology and Medicine, Vol. 42/8, Pergamon, Bergama, https://doi.org/10.1016/J.FREERADBIOMED.2007.01.020.

Shirai, K. et al. (2006), "Differential effects of x-irradiation on immature and mature hippocampal neurons in vitro", Neuroscience Letters, Vol. 399/1–2, Elsevier, Amsterdam, https://doi.org/10.1016/j.neulet.2006.01.048.

Shirai, K. et al. (2013), "X Irradiation Changes Dendritic Spine Morphology and Density through Reduction of Cytoskeletal Proteins in Mature Neurons", Radiation Research, Vol. 179/6, Allen Press, Lawrence, https://doi.org/10.1667/RR3098.1.

Takahashi, H. et al. (2003), "Drebrin-Dependent Actin Clustering in Dendritic Filopodia Governs Synaptic Targeting of Postsynaptic Density-95 and Dendritic Spine Morphogenesis", The Journal of
Tiller-Borcich, J. K. et al. (1987), "Pathology of Delayed Radiation Brain Damage: An Experimental Canine Model", Radiation Research, Vol. 110/2, Allen Press, Lawrence, https://doi.org/10.2307/3576896.

Whoolery, C. W. et al. (2017), "Whole-body exposure to 28Si-radiation dose-dependently disrupts dentate gyrus neurogenesis and proliferation in the short term and new neuron survival and contextual fear conditioning in the long term", Radiation Research, Vol. 188/5, Radiation Research Society, https://doi.org/10.1667/RR14797.1.
# Relationship: 2838 – Energy Deposition leads to Impairment, Learning and memory

## AOPs Referencing Relationship

<table><thead><tr><td>AOP Name</td><td>Adjacency</td><td>Weight of Evidence</td><td>Quantitative Understanding</td></tr></thead><tbody><tr><td>AOP: 483 - Deposition of Energy Leading to Learning and Memory Impairment</td><td>non-adjacent</td><td>Moderate</td><td>Low</td></tr></tbody></table>

## Evidence Supporting Applicability of this Relationship

Evidence for this relationship comes from human, rat, and mouse models, with a large amount of evidence in mice and rats. There is in vivo evidence in both male and female animals, with a lot of evidence in males. This relationship has been shown in adult animals in many studies.

## Taxonomic Applicability

<table><thead><tr><td>Term</td><td>Scientific Term</td><td>Evidence</td><td>Link</td></tr></thead><tbody><tr><td>human</td><td>Homo sapiens</td><td>Low</td><td>NCBI</td></tr><tr><td>mouse</td><td>Mus musculus</td><td>High</td><td>NCBI</td></tr><tr><td>rat</td><td>Rattus norvegicus</td><td>High</td><td>NCBI</td></tr></tbody></table>

## Life Stage Applicability

<table><thead><tr><td>Term</td><td>Evidence</td></tr></thead><tbody><tr><td>Adult</td><td>High</td></tr><tr><td>Old Age</td><td>Low</td></tr><tr><td>Juvenile</td><td>Low</td></tr></tbody></table>

## Sex Applicability

<table><thead><tr><td>Sex</td><td>Evidence</td></tr></thead><tbody><tr><td>Male</td><td>High</td></tr><tr><td>Female</td><td>Low</td></tr></tbody></table>

## Key Event Relationship Description

Deposition of energy from ionizing radiation (IR) can induce biological changes within living systems (UNSCEAR, 1982). The amount of IR absorbed and consequently the amount of damage ensued is quantified by the linear energy transfer (LET) of the radiation. Low-LET radiation consists of electromagnetic ionizing radiation such as X- and gamma rays, as well as protons that deposit smaller amounts of energy, whereas high-LET radiation deposits large amounts of energy and includes heavy ions, alpha particles and high-energy and neutrons. Therefore, high-LET radiation produces dense ionization while low-LET radiation induces sparse ionization events where energy is exponentially absorbed by tissues.

Deposition of energy can lead to reduced cognitive function related to learning and memory. Impaired learning can be seen as diminished ability to create new associative or non-associative relationships, whereas impaired memory consists of reduced ability to establish sensory, short-term or long-term memories (Desai et al., 2022; Kiffer et al., 2019b). Multiple brain areas are involved in learning and memory processes, with the most well-known occurring in the hippocampal region, as well as the amygdala, the prefrontal cortex, the basal ganglia, and other areas of the neocortex. These regions of the brain have been shown to be affected by deposition of energy (Cucinotta et al., 2014; Desai et al., 2022; NCRP Commentary, 2016).

Following deposition of energy, the process begins at the macromolecular level from direct damage to neurons and glial cells and via the generation of oxidative stress and promotion of neuroinflammatory environments in the central nervous system (CNS) (Mhatre et al., 2022; Lalkovičová et al., 2022). The altered cellular environment caused by a deposition of energy can
impact the functions of both neurons and glial cells, which can promote a persistent pro-inflammatory response, reduced neurogenesis, reduced dendritic spine lengths and density, and the inhibition of neuronal connectivity and synaptic activity (Hladik & Tapio, 2016; Cekanaviciute et al., 2018). In vivo studies link structural and functional changes in neurons and glial cells to a decreased ability to complete cognitive assessments that test various domains of learning and memory.

## Evidence Supporting this KER

The strategy for collating the evidence to support the relationship is described in Kozbenko et al 2022. Briefly, a scoping review methodology was used to prioritize studies based on a population, exposure, outcome, endpoint statement.

Overall Weight of Evidence: Moderate

## Biological Plausibility

The biological rationale linking deposition of energy to impaired learning and memory is strongly supported in the literature, as reported by several review articles published on the subject (Desai et al., 2022; Kiffer et al., 2019b; Pasqual et al., 2021; NCRP, 2016; Collett et al., 2020; Cucinotta et al., 2014; Hladik & Tapio, 2016; Cekanaviciute et al., 2018; Mhatre et al., 2022; Lalkovičová et al., 2022; Greene-Schloesser et al., 2012; Turnquist et al., 2020; Katsura et al., 2021). It is well established that radiation exposure leads to both acute and chronic elevation in reactive oxygen species (ROS), and this subsequently mediates cell signaling within mature neurons, neural stem cells and glial cells (NCRP, 2016; Mhatre et al., 2022). ROS acts as a second messenger to activate microglia and astrocytes via redox-responsive transcription factor-mediated molecular signaling pathways, and once these glial cells are activated, they can adopt a pro-inflammatory morphology and thus release pro-inflammatory mediators (Hladik & Tapio, 2016; Cucinotta et al., 2014; Collett et al., 2020). Pro-inflammatory mediators such as cytokines mediate the immune response through ligand binding to cell surface receptors that can activate signaling cascades such as JAK-STAT or MAPK pathways to produce or recruit more cytokines (Mousa & Bakhiet, 2013; Prieto & Cotman, 2018).

Once these inflammatory reactions are initiated, the radiation-activated microglia can alter the functions of neurons. Structurally, the neuron is comprised of the cell body, dendrites, axon, and axon terminals (Lodish et al., 2000). Neurons communicate by electrical and chemical signaling via synapses, where axons from the presynaptic neuron interface with the dendritic arms of the postsynaptic neuron. Deposition of energy inhibits neuronal connectivity and synaptic activity through the loss of dendritic spines as well as dendrite length and branching (Cekanaviciute et al., 2018; Jandial et al., 2018). Overexpression of pro-inflammatory mediators disrupts the integrity of neurons through increased necrosis and demyelination, decreased neurogenesis, decreased neural stem cell proliferation and decreased synaptic complexity (Hladik & Tapio, 2016; Cekanaviciute et al., 2018; Lalkovičová et al., 2022). Together, these radiation-induced neuronal damage and persistent neuroinflammation alter learning and memory capabilities as evidenced by behavioral changes from in vivo studies.

## Empirical Evidence

There is strong evidence supporting the connection between the deposition of energy leading to impaired learning and memory. The evidence was gathered from studies using in vivo rodent models and human studies. The applied stressors ranged from 0.5 cGy to 40 Gy and included heavy ions (e.g., 28Si, 16O, 56Fe, 48Ti), X-rays, gamma rays, protons, and alpha particles. Impaired learning and memory in rodents were measured through various behavioral tests that assessed several cognitive aspects such as short-term memory, long-term memory, recognition memory, spatial memory, working memory, declarative memory, associative learning, discrimination, and reversal learning (Britten et al., 2018; Rabin et al., 2014; Lonart et al., 2012; Britten et al., 2012; Bellone et al., 2015; Parihar et al., 2016; Rabin et al., 2012; Krukowski et al., 2018a; Forbes et al., 2014; Gan et al., 2019; Manda et al., 2007a; Acharya et al., 2016; Impey et al., 2016; Parihar et al., 2018; Belarbi et al., 2013; Krukowski et al., 2018b; Jewell et al., 2018; Rabin et al., 2015; Parihar et al., 2015; Kiffer et al., 2019a
; Hodges et al., 1998). Additionally in humans, cognitive test scores from the Swedish military
enlistment exam of 18-year-old men, which evaluates general instruction, concept discrimination,
technical comprehension, and spatial recognition, were used to evaluate the potential impact of beta
ray, gamma ray, or X-ray exposures from radiotherapy treatment for cutaneous haemangioma when
they were infants (18 months old or earlier) (Hall et al., 2004).

Dose Concordance

Many studies demonstrate dose concordance relating to deposition of emerging and impaired learning and memory. NOR is a behavioral assay that is used to evaluate recognition memory by measuring the time spent with a novel object instead of a familiar one after habituation and training with the familiar object (Cekanaviciute et al., 2018). Mice irradiated with 9 Gy X-rays showed a DI (discrimination index; tendency to explore novel instead of familiar objects/locations) of -13 compared to the control of 23, demonstrating impaired memory after irradiation (Acharya et al., 2016). Another study in rats reported a reduction in the discrimination ratio (DR) from 0.5 to 0.2 after 40 Gy X-rays, also suggesting reduced learning and memory after radiation exposure (Forbes et al., 2014). However, this was only observed in 3-month-old rats. Mice irradiated with 0.05 or 0.3 Gy of either 16O and 48Ti particles showed a dose- and particle size-dependent decrease in the DI for NOR (Parihar et al., 2015; Parihar et al., 2016). Another study with heavy ions also reported impairments in novel object recognition after 0.1 Gy and 0.4 Gy of 56Fe ion irradiation, but not after 0.2 Gy of 56Fe ion irradiation (Impey et al., 2016). Memory was also inhibited in mice after 4He particle irradiation at 0.5 Gy, but not after 4He particle irradiation at 0.15 Gy or 1 Gy after being tested using NOR (Krukowski et al., 2018b). In a mixed ion beam study using the galactic cosmic ray (GCR) simulator, NOR was impaired after 0.15 Gy and 0.5 Gy of irradiation in males only, with no reduction in performance reported in female mice (Krukowski et al., 2018a). In contrast, results from a separate study suggest that there is an impact on female mice, as female mice irradiated with 16O particles showed dose-dependent decreases in the DR from 22 (control) to -2 (0.1 Gy) and -5 (0.25 Gy) for NOR (Kiffer et al., 2019a). Memory assessed with NOR was also impaired in rats exposed to various particles (protons, carbon, oxygen, silicon, titanium and iron ions) with doses from 0.1 to 2 Gy (Rabin et al., 2014). Rats irradiated with 0.25 Gy 56Fe or 0.05 Gy 16O particles showed attenuated memory, but not learning, measured with NOR (Rabin et al., 2015).

Object in Place (OiP), a similar test to NOR where the same objects are used but the locations of one or more of the objects is changed, is also used to assess memory (Cekanaviciute et al., 2018). Mice irradiated with 9 Gy X-rays showed a DI of -3 compared to the control of 38 suggesting impaired memory (Acharya et al., 2016). Mice irradiated with 16O particles showed a dose-dependent decrease in the DI for NOR at 0.05 or 0.3 Gy, while 48Ti irradiation also resulted in significantly decreased DI at both 0.05 and 0.3 Gy (Parihar et al., 2015; Parihar et al., 2016). After 0.05 and 0.3 Gy 4He irradiation, mice also showed reduced performance in the OiP test suggesting impaired memory (Parihar et al., 2018).

A few studies used the multistage attentional set shifting (ATSET) test, which measures the ability to relearn cues over various schedules of reinforcement (Heisler et al., 2015). During ATSET, simple discrimination (SD) was inhibited by 0.05, 0.15 and 0.2 Gy of 28Si particles in rats, shown by increased latency to complete the task and a 20 to 30 percentage point decrease in completion of various stages (Britten et al., 2018). Of rats irradiated with 0.2 Gy 56Fe particles, 2 of 11 completed all stages, while 8 of 11 control rats completed all stages (Lonart et al., 2012). In addition, the same study (Lonart et al., 2012) demonstrated that irradiated rats required significantly more attempts to complete the SD stage. The number of attempts for compound discrimination (CD) was increased about 2-fold from 0.01 to 0.15 Gy in rats irradiated with 56Fe particles (Jewell et al., 2018). There was also a dose-dependent decrease in the percent of rats that failed at least 1 stage. Similarly, rats took more trial (2X) to complete the CD stage after 5 cGy of 48Ti (Parihar et al., 2016).

Fear conditioning (FC) and fear extinction (FE) are used to assess learning and memory. FC measures the learned fear response to an adverse event, while FE measures the dissociation of this response to the adverse event (Parihar et al., 2016). Mice tested with fear conditioning after 9 Gy X-rays spent
19% of the time frozen, while control mice spent 43% of the time frozen during the context test (Acharya et al., 2016). Mice irradiated with 0.3 Gy 48Ti showed similar levels of FC to controls, but FE was inhibited in irradiated mice as they were unable to abolish fear memory (Parihar et al., 2016). This also occurred for 0.05 Gy 4He particles (Parihar et al., 2018).

Various maze tests were performed to assess learning and memory. During an MWM test, control mice took 246 cm to reach the hidden platform, while 10 Gy-irradiated mice took 392 cm (Belarbi et al., 2013). A water maze did not show initial differences in learning and memory after 0.5 Gy protons, but reversal learning, where mice had to forget what they had previously learned, showed 1.6-fold increased swim distance to the hidden platform in irradiated mice (Bellone et al., 2015). Rats irradiated with X-rays (13 Gy) and 56Fe particles (0.2, 0.4 and 0.6 Gy) showed increased escape latency compared to controls (Britten et al., 2012). A Hebb-Williams maze after rats were irradiated with 6 Gy gamma rays showed significant decreases in learning and memory as well (Manda et al., 2007a). After irradiation, rats exposed to 25 Gy had significant impairments in both the T-maze, a measure of spatial working memory, and water maze tests, while rats exposed to lower dose of 20 Gy, had significant impairment of working memory in the T-maze (Hodges et al., 1998).

A Swedish cohort study by Hall et al. (2004) showed that exposure to β-rays, gamma rays and X-ray doses of 1 to 250 mGy for treatment of cutaneous hemangioma during infancy was related to reduced test scores approximately 18 years later, including tests of concept discrimination (P=0.03), general instruction (P=0.03) and technical comprehension (P=0.003). In mice, reduced performance on temporal order (TO) tasks, where short-term memory is assessed through the animals' preference to two sets of objects, was reported after 0.05 and 0.3 Gy of 16O, 48Ti and 4He particle irradiation (Parihar et al., 2016; Parihar et al., 2018). A three-chamber social approach task, which assesses social memory, was impaired in male mice after 0.5 Gy of exposure to the GCR simulator compared with animals exposed to 0.15 Gy of GCR simulator. However, there were no significant differences in TO in the exposed animals compared to unexposed controls (Krukowski et al., 2018a). Performance on operant responding, which reflects the cortex' ability to organize processes was assessed in rats. Performance on this test was reduced after 0.25, 0.5, 1 and 2 Gy 56Fe ion exposure, suggesting alterations in learning and memory (Rabin et al., 2012). However, there was no consistent trend across doses (Rabin et al., 2012).

Furthermore, a meta-analysis identified several studies that showed a risk of developing central nervous diseases such as Alzheimer's and dementia after exposure to low-to-moderate doses of ionizing radiation in adulthood. The various studies investigated CNS diseases in relation to occupational exposure, environmental exposure, those exposed for medical purposes, Chernobyl cleanup workers and Japanese Atomic Bomb Survivors with dose ranges <1 Gy. Results from the meta-analysis suggest that there is no increase in standardized mortality ratio (SMR) from diseases of the nervous system when comparing the radiation exposed cohorts to the general population. However, there was a positive and significant excess relative risk from ionizing radiation for Parkinson's disease (Azizova et al., 2020; Lopes et al., 2022).

## Time Concordance

Novel object recognition (NOR) was used in multiple studies to assess recognition memory at varying time points following energy deposition. In many cases, the deposition of energy by radiation led to impaired recognition memory in the animal models studied. A decrease in DI indicates a reduction in recognition memory. At 5 to 6 weeks after 9 Gy irradiation, mice had decreased DI for NOR (Acharya et al., 2016). Similarly, at 6 weeks after 48Ti (0.05 and 0.30 Gy) and 16O (0.30 Gy) irradiation, the DI for NOR decreased significantly in male mice (Parihar et al., 2015). After exposure to 5 and 30 cGy of 48Ti, cognitive impairment was greater after 24 weeks than 12 weeks as evidenced by reduced NOR DI for both doses of 48Ti (Parihar et al., 2016). Mice at 90 days post helium exposure or 45 days post GCR simulator radiation (0.15 or 0.50 Gy) both demonstrated memory impairments as mice were unable to distinguish novel and familiar objects (Krukowski et al., 2018a; Krukowski et al., 2018b). After 2 weeks of exploring, mice previously irradiated with 0.1 or 0.4 Gy showed impaired object recognition; however, at the 20-week time-point, no impairment was indicated following 0.1, 0.2 or
0.4 Gy of 56Fe (Impey et al., 2016). NOR performance was impaired in mice at 2- and 4-months post 16O irradiation, 11 months post 12C irradiation, at 3- and 12-months post 56Fe exposure and at 7- and 17-months post 48Ti irradiation. As well, NOR was impaired 4, 5, 9, 10 and 13 months after 28Si exposure, with the lowest performance occurring at 9 months (Rabin et al., 2014). Mice demonstrated impaired memory 270 days after 16O radiation at 0.01 Gy or 0.25 Gy (Kiffer et al., 2019a). Four and 13 months after proton exposure, NOR was impaired in rats (Rabin et al., 2014). Also in rats, memory ability decreased 18 h after 56Fe and 16O exposure but no significant changes in learning ability by NOR were found after 48 h (Rabin et al., 2015). Months after 40 Gy X-ray irradiation, rats showed differences in NOR results by age. Rats that were 3 months old at the time of irradiation, showed a decrease in NOR 3 months post irradiation, while no changes were observed in older ages and later timepoints (Forbes et al., 2014). No changes in novel object location (NOL) were observed for rats between 6 and 15 months of age (Forbes et al., 2014).

To study spatial learning and memory, mice and rat models were subjected to various maze tests. Proton-irradiated mice tested with Barnes maze and water maze did not show impairment 3 months after irradiation (Bellone et al., 2015). However, 3 months after exposure to 13 Gy of X-rays, rats became less capable of escaping the Barnes maze and demonstrated increased escape latency (Britten et al., 2012). After 6 months, mice reached the platform with a longer distance during the reversal learning test (Bellone et al., 2015). Rats exposed to 0, 8 and 10 Gy of X-rays experienced no impairments in spatial memory, while those exposed to 0.2, 0.4 and 0.6 Gy 56Fe demonstrated increased relative escape latency over the 3-day testing periods (Britten et al., 2012). Cognitive changes in spatial learning or memory were observed in the water maze after 2 weeks, but not at 20 weeks (Impey et al., 2016). Mice studied in a water maze 1 year following 4He irradiation (0.05 and 0.30 Gy) showed impaired cognitive flexibility and memory retrieval (Parihar et al., 2018). Mice previously irradiated with 6 Gy gamma rays needed a 1.7-fold increase in time to reach the goal of the Hebb-William maze by day 30 post-irradiation, while in the control, the time required decreased 0.2-fold by day 30 (Manda et al., 2007a). Irradiated mice 9 months after 16O irradiation at 0.1 and 0.25 Gy had no difference in positive discrimination ratio during a Y-maze test, a measure spatial working memory, compared with control mice, indicating that radiation had no effect on memory 9 months post-irradiation (Kiffer et al., 2019a). Rats irradiated at 20 Gy and 25 Gy experienced deficits in T-maze testing 35 weeks post irradiation and at 44 weeks, rats exposed to 25 Gy reflected working memory deficits assessed by the water maze test (Hodges et al., 1998).

OiP used in multiple studies showed impairment in discrimination of objects after irradiation. Mice 5 to 6 weeks after 9 Gy irradiation showed decreased OiP DI (Acharya et al., 2016). Similarly, 6 weeks after charged particle exposure, the DI for OiP in male mice decreased to a greater extent following 48Ti irradiation compared to 16O irradiation (Parihar et al., 2015). As well, a greater change was observed after 0.30 Gy of 16O irradiation compared with 0.05 Gy of 16O irradiation after 6 weeks, indicating higher impairment at the higher dose (Parihar et al., 2015). In a similar experiment to Parihar et al. (2015), the OiP DI decreased after 0.30 Gy of 48Ti irradiation at week 12, in addition to DI decrease at 24 weeks after 0.30 Gy of 16O irradiation and 0.05 Gy of 48Ti irradiation. Hence, cognitive impairment was greater after 24 weeks than 12 weeks (Parihar et al., 2016). 4He particle irradiation of mice led to long-term recognition memory impairments at 6, 15 and 52 weeks post-radiation (Parihar et al., 2018).

Fear conditioning and fear extinction of previously irradiated mice were studied at different timepoints after irradiation. Mice irradiated with 0.3 Gy and 0.05 Gy spent more time freezing their motion than the control mice at 24 weeks and 1 year, respectively, following irradiation (Parihar et al., 2016; Parihar et al., 2018). Mice irradiated with 9 Gy X-rays spent less time frozen during FC, measured after 5-6 weeks (Acharya et al., 2016). FE was attenuated in mice after 0.3 Gy of 48Ti irradiation after 24 weeks post-irradiation (Parihar et al., 2016). The same was found after 0.05 Gy of 4He exposure 1-year post-irradiation (Parihar et al., 2018).

ATSET was used to assess simple and compound discrimination, reversal learning and set shifting, among others. Only 2 out of 11 of the irradiated rats (0.20 Gy of 56Fe) completed all the paradigms
90 days after radiation compared to 8 out 11 unirradiated rats that completed all paradigms (Lonart et al., 2012). At 90-days post-irradiation, irradiated rats required more trial attempts to complete the simple discrimination stage compared to the control rats (Lonart et al., 2012). Rats took 2-fold more tries to complete the CD stage 12 weeks after radiation (Parihar et al., 2016).

In irradiated mice, temporal order DI decreased at 12 and 24 weeks for all tested doses (0.05 or 0.30 Gy of 16O or 48Ti), demonstrating cognitive impairment (Parihar et al., 2016). Meanwhile, Parihar et al. (2018) found impaired TO memory for 6-, 15- and 52-weeks post 4He particle irradiation (Parihar et al., 2018). Male mice had significantly impaired social memory in the three-chamber social approach task at day 45 following exposure to 0.50 Gy of the GCR simulator (Krukowski et al., 2018a). Operant responding as a cognitive function test was used on 56Fe irradiated rats who showed decreased cognitive function at 2, 4-8, 10-, and 15-months post-irradiation (Rabin et al., 2012). Swedish men who were irradiated under 18 months of age were found to have cognitive impairments following reduced test scores for technical comprehension, concept discrimination and general instruction at 18 and 19 years old (Hall et al., 2004).

## Essentiality

As deposition of energy is a physical stressor, it cannot be blocked by chemicals although it can be shielded. Further research is required to determine the effect of shielding radiation on learning and memory. Since deposited energy initiates events immediately, the removal of deposited energy also supports the essentiality of the key event. Studies that do not deposit energy are observed to have no downstream effects. However, impaired learning and memory can occur in response to ionizing radiation, although in the absence of this energy deposition, compromised cognitive abilities would occur with aging, or if predisposed to neurodegenerative diseases such as Alzheimer's (Lindsay et al., 2002).

## Uncertainties and Inconsistencies

* Most of the evidence for this KER is supported using in vivo rodent models; therefore, there is still a knowledge gap on how radiation exposure realistically alters the brains of other species, such as humans, to lead to impaired learning and memory (Desai et al., 2022). Additionally, further research is needed to gain a better understanding on the sex differences in behavioral effects after radiation exposure (Kiffer et al., 2019b).

* Belarbi et al. (2013) did not find any changes in NOR performance after 10 Gy gamma rays.

* Forbes et al. (2014) showed impaired ability during NOR tests, but not during NOL tests after 40 Gy X-rays.

* Kiffer et al. (2019a) showed impaired ability during NOR tests, but not during Y-maze tests after 0.1 and 0.25 Gy 16O particles.

* Miry et al. (2021) showed impaired hippocampal-dependent learning and memory 2 months after 10, 50 and 100 cGy 56Fe exposure, but by 6 months post-exposure, deficits in spatial learning were no longer observed in irradiated mice. Instead, enhanced spatial learning was observed at 12- and 20-months post-exposure.

## Known modulating factors

<table><thead><tr><td>Modulating Factor</td><td>Details</td><td>Effects on the KER</td><td>References</td></tr></thead><tbody><tr><td>Drug</td><td>PLX5622-1200ppm (PLX) diet that contains a CSF1-R (colony stimulating factor 1 receptor) inhibitor that induces depletion of microglia within 3 days.</td><td>The PLX diet was able to rescue the short and long-term memory impairments due to radiation exposure.</td><td>Krukowski et al., 2018b; Acharya et al., 2016</td></tr><tr><td>Drug</td><td>Fluoxetine treatment – an antidepressant that belongs to the selective serotonin reuptake inhibitor (SSRI) class.</td><td>Fluoxetine was able to attenuate the learning and memory defects in mice that were subjected to radiation.</td><td>Gan et al., 2019</td></tr></tbody></table>
<table><tr><td>Genotype</td><td>CCR2 (chemokine C-C motif receptor 2) knockout. CCR2 is involved in peripheral macrophage infiltration at the sites of injury in the CNS.</td><td>CCR2 deficiency was able to prevent the cognitive impairments induced by cranial radiation as shown by improvements in the MWM test.</td><td>Belarbi et al., 2013</td></tr><tr><td>Drug</td><td>Treatment with α-lipoic acid (LA) as it has antioxidant properties.</td><td>LA-treated mice did not show any significant decline in spatial memory post-irradiation.</td><td>Manda et al., 2007b</td></tr><tr><td>Sex</td><td>Male and female mice responded differently to NOR, three chamber social approach and open field task following irradiation.</td><td>Male animals generally demonstrated memory impairment after irradiation.</td><td>Krukowski et al., 2018a</td></tr><tr><td>Drug</td><td>Treatment with melatonin as it is involved in many physiological processes and has antioxidant properties.</td><td>Pre-treatment with melatonin showed significant protection against impairment in learning ability. Melatonin was found to reduce the time taken to reach the goal in a Hebb-Williams maze compared to irradiation.</td><td>Manda et al., 2007a</td></tr><tr><td>Various modulating factors</td><td>Commonly applied countermeasures include:<ul><li>Those targeting the reduction of oxidative stress (ie. Pharmaceutical antioxidants, nutritional antioxidants)</li><li>Decreasing DNA damage (ie. Overexpression of BMI1 gene to accelerate DNA repair)</li><li>Enhancing cell survival (ie. Inhibition of p53-induced apoptosis)</li><li>Reducing inflammation (ie. Pharmaceutically blocking pro-inflammatory cytokine/chemokine signaling)</li><li>Limiting tissue damage and increasing repair (ie. Cell transplants)</li></ul></td><td>Various approaches to CNS radioprotection have been utilized as either primarily protective (administered prior to irradiation) or mitigative (administered after irradiation). These countermeasures have been found to limit the harmful effects of radiation exposure.</td><td>Pariset et al., 2021</td></tr><tr><td>Stress</td><td>Psychological Stress</td><td>Psychological stress related to perceived risk of radiation exposure can also impact learning and memory.</td><td>Collett et al., 2020</td></tr></table>

## Quantitative Understanding of the Linkage

The table below provides some representative examples of quantitative linkages between the two key events. It was difficult to identify a general trend across all the studies due to differences in experimental design and reporting of the data. All data is statistically significant unless otherwise stated.

### Dose Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Hodges et al., 1998</td><td>In vivo. Male Sprague-Dawley Rats were irradiated with X-rays at 0, 20 Gy or 25 Gy at 1.4 Gy/min. Rats were tested from 26 weeks to 44 weeks after irradiation using the T maze and water maze to assess working memory and brain damage induced by radiation.</td><td>Rats irradiated with 25 Gy, showed impairments in all tests and rats irradiated with 20 Gy were not as impaired in the water maze learning and had no impairment in water maze working memory. Rats irradiated with 20 Gy had significant impairment of working memory in the T-maze.</td></tr><tr><td>Hall et al., 2004</td><td>In vivo. A Swedish cohort study that analyzed military tests of learning ability, logical reasoning and spatial recognition of 2211 men 17-18 years after β-ray, 26Ra gamma ray or X-ray radiation for cutaneous hemangioma before the age of 18 months from 1930-1959.</td><td>Test scores for concept discrimination and general instruction as well as technical comprehension decreased in a dose-dependent manner from 0 to 250 mGy. Spatial recognition did not significantly change.</td></tr><tr><td>Acharya et al., 2016</td><td>In vivo. C57Bl/6J mice were irradiated with 9 Gy X-rays and novel object recognition (NOR), OiP and FC were measured. The DI was calculated from exploration times in familiar and novel locations. Percent of time spent freezing was used to determine FC.</td><td>The DI for NOR was 23 without irradiation and -13 with 9 Gy. The DI for OiP was 38 without irradiation and -3 with 9 Gy. Mice without irradiation spent 43% of the time frozen and mice with 9 Gy spent 19% of the time frozen during the context test.</td></tr><tr><td>Belarbi et al., 2013</td><td>In vivo. C57Bl/6J mice were irradiated with 10 Gy 137Cs gamma rays. NOR and MWM were used to determine learning and memory impairment.</td><td>NOR did not show any significant differences between control and 10 Gy irradiated groups. In MWM, control mice traveled 246 cm to the hidden platform, while 10 Gy irradiated mice traveled 392 cm to the hidden platform.</td></tr></tbody></table>
<table>
  <tr>
    <td>Bellone et al., 2015</td>
    <td>In vivo. Male B6C3F1/J mice were irradiated with 150 MeV protons at 0.5 Gy (1.5-2.5 Gy/min). A water maze was used to assess learning and memory.</td>
    <td>No changes were found initially during the water maze test, but irradiated mice took a 1.6-fold longer swim distance to reach the platform during the reversal learning phase.</td>
  </tr>
  <tr>
    <td>Britten et al., 2012</td>
    <td>In vivo. Wistar rats were irradiated with 125 kVp X-rays or 1 GeV/u 56Fe ions to study the effect of low dose exposure in the spatial learning ability of rats. Rats were tested 3 months after irradiation using the Barnes maze.</td>
    <td>13 Gy X-ray irradiated rats and rats exposed to all doses of 56Fe ions studied (0.2, 0.4 and 0.6 Gy) demonstrated a higher relative escape latency of 1.8-fold and ~2.5-fold, respectively, compared to the control group.</td>
  </tr>
  <tr>
    <td>Britten et al., 2018</td>
    <td>In vivo. Wistar rats were exposed to heavy ions, 600 MeV/n 28Si. Attentional set shifting (ATSET) was used to assess various cognitive processes.</td>
    <td>28Si irradiation of rats significantly reduced their ability to complete simple discrimination. Rats exposed to 0.05 Gy, 0.15 Gy and 0.20 Gy had a lower percentage stage completion of simple discrimination compared to 0 Gy rats (60-70% completion compared to 90%). 0.15 Gy irradiated rats also had a lower completion rate of compound discrimination compared to 0 Gy rats.</td>
  </tr>
  <tr>
    <td>Lonart et al., 2012</td>
    <td>In vivo. Male Wistar rats were irradiated with 56Fe ions. ATSET test was used to assess simple and compound discrimination, reversal learning and set-shifting, among others.</td>
    <td>8 of 11 unirradiated rats completed all the paradigms, while only 2 of 11 irradiated rats (0.20 Gy) completed all the paradigms. Irradiated rats required more trial attempts to complete the simple discrimination stage compared to unirradiated rats.</td>
  </tr>
  <tr>
    <td>Forbes et al., 2014</td>
    <td>In vivo. Male FxBN rats were irradiated with X-rays at 40 Gy from 2 fractions of 5 Gy per week over 4 weeks (each fraction at 1.25 Gy/min). Learning and memory were assessed using NOR and NOL.</td>
    <td>40 Gy significantly reduced the DR of 3-month-old rats during NOR from 0.5 to 0.2. No changes in NOL were observed, and no changes at other ages were observed.</td>
  </tr>
  <tr>
    <td>Parihar et al., 2015</td>
    <td>In vivo. Male Thy1-EGFP transgenic mice were irradiated with charged particles (16O and 48Ti) at 600 MeV/amu (0.5 to 1.0 Gy/min). NOR and OiP tests were done to assess learning and memory.</td>
    <td>NOR:<br>The control group DI was 39. The DI was 25 after 5 cGy 16O, 10 after 30 cGy 16O, 2 after 5 cGy 48Ti and -4 after 30 cGy 48Ti.<br>OiP:<br>The control group DI was 32. The DI was 29 after 5 cGy 16O, -4 after 30 cGy 16O, -8 after 5 cGy 48Ti and -3 after 30 cGy 48Ti.</td>
  </tr>
  <tr>
    <td>Parihar et al., 2016</td>
    <td>In vivo. Male Thy1-EGFP transgenic mice were irradiated with charged particles (16O and 48Ti) at 600 MeV/amu (0.05 to 0.25 Gy/min). NOR, OiP, TO and FE tests were done to assess learning and memory. Wistar rats were used in the ATSET test.</td>
    <td>Impairment in cognitive ability was observed at both 5 and 30 cGy. The impairment was typically greater in 48Ti than 16O. The DI was between 20 and 40 for all controls and was reduced to less than 0 after many of the different radiation treatments.</td>
  </tr>
  <tr>
    <td>Parihar et al., 2018</td>
    <td>In vivo. Male C57BL/6J mice were irradiated with 4He particles (400 MeV/n) at 5 cGy/min. Learning and memory were assessed using OiP, TO and FE.</td>
    <td>Cognitive tests showed impaired learning and memory at both 5 and 30 cGy (except FE which was only tested at 5 cGy). For example, control mice spent 2-fold more time exploring novel objects than familiar objects, while irradiated rats did not spend significantly different time exploring novel objects. Impairment at 30 cGy was not significantly different than at 5 cGy.</td>
  </tr>
  <tr>
    <td>Impey et al., 2016</td>
    <td>In vivo. Male C57BL/6J mice were irradiated with 600 MeV 56Fe ions at 0.1, 0.2 or 0.4 Gy and various dose rates from 0.25 to 0.36 Gy/min. NOR and a water maze were used to determine learning and memory ability.</td>
    <td>Transiently, mice with 0.1 and 0.4 Gy spent more time exploring the familiar object and less exploring the novel object. No differences were observed in the water maze test.</td>
  </tr>
  <tr>
    <td>Jewell et al., 2018</td>
    <td>In vivo. Male Wistar rats were irradiated with 56Fe particles (600 MeV/n) at 1 and 3 cGy (2 cGy/min), or 5, 10 and 15 cGy (5 cGy/min). ATSET was performed to assess various cognitive processes.</td>
    <td>At every dose, the number of attempts for CD was significantly increased, with a maximum of 2-fold. Various doses were also able to significantly increase the number of attempts for SD, CD reversal and intra-dimensional shifting (IDS). Significant increases in time to complete the test were sparse. However, doses of 3 cGy and above showed nonsignificant increases in latency in almost all tests.</td>
  </tr>
  <tr>
    <td>Krukowski et al., 2018b</td>
    <td>In vivo. C57BL/6J wild type mice were exposed to alpha particles at 0, 15, 50 or 100 cGy (16.37 cGy/min). NOR was used as an assay for behavioral analysis at 90 days post-irradiation.</td>
    <td>After 50 cGy exposure, animals spent similar time exploring the familiar and novel object, indicating memory impairment.</td>
  </tr>
  <tr>
    <td>Krukowski et al., 2018a</td>
    <td>In vivo. Male and female C57BL/6J wild type mice were exposed to 0, 15 or 50 cGy of GCR simulator (2.54 cGy/min). NOR assay was used to study recognition memory. A three-chamber social approach task determined social behavior and social memory.</td>
    <td>NOR:<br>Male mice demonstrated memory impairment when exposed to 15 or 50 cGy.<br>Three-chamber social approach task:<br>Male mice exposed to 50 cGy showed impairments in social memory compared to the 15 cGy group.</td>
  </tr>
  <tr>
    <td>Kiffer et al., 2019a</td>
    <td>In vivo. Female C57BL/6 mice were irradiated with 16O particles (600 MeV/n) at 0.1 or 0.25 Gy (18 to 33 cGy/min). A Y-maze was performed to measure spatial memory and NOR was performed to measure non-spatial declarative memory.</td>
    <td>Y-maze:<br>No changes were observed in irradiated mice.<br>NOR:<br>The DR was 22 for the control, -2 for 0.1 Gy and -5 for 0.25 Gy. Both changes were significant.</td>
  </tr>
</table>
<table><tr><td>Manda et al., 2007a</td><td>In vivo. Male Swiss albino mice were irradiated with 6 Gy 60Co gamma rays. Hebb-Williams maze was used to measure learning ability and spatial working memory continuously for 30 days after radiation exposure.</td><td>Irradiated mice took approximately 8 times longer to reach their goals and their learning ability declined after radiation exposure compared to controls and mice pre-treated with melatonin.</td></tr><tr><td>Rabin et al., 2012</td><td>In vivo. Male Fischer 344 rats were irradiated with various doses (50 to 100 cGy/min) of 56Fe particles (1000 MeV/n). Operant responding was used to test cognitive function. Rats were trained to press a lever to receive food at various reinforcement schedules.</td><td>Doses of 25, 50, 150 and 200 cGy influenced cognitive function. However, there was no significant trend related to dose.</td></tr><tr><td>Rabin et al., 2014</td><td>In vivo. Male Sprague-Dawley rats were irradiated with various types of ionizing radiation (5 to 100 cGy/min so the time did not exceed 3 to 4 min). LET ranged from 0.22 (protons) to 181 (56Fe) keV/µM. The dose ranged from 0.1 to 200 cGy. The radiation types used were 16O (600 and 1000 MeV/n), 12C (290 MeV/n), 28Si (380, 600 and 1000 MeV/n), 48Ti (1100 MeV/n), 56Fe (600 MeV/n) and protons (1000 MeV/n). NOR was performed to assess learning and memory.</td><td>In most particles studied, a lower or equal dose to that of younger rats was needed to disrupt NOR and cognitive performance in older rat subjects. Recognition memory performance was disrupted by a dose 10% - 50% less in older rats than younger rats. Older rats irradiated with 380 MeV/n 28Si particles showed a recognition memory disruption at a lower dose (0.005 Gy in older rats compared to 0.1 Gy in younger rats). Whereas higher dose was necessary to disrupt recognition memory in older rats exposed to 1000 MeV/n 28Si particles compared to the younger rats (0.5 Gy in older rats compared to 0.25 Gy in younger rats). Following 28Si radiation, memory was impaired at every dose from 0.1 to 2 Gy.</td></tr><tr><td>Rabin et al., 2015</td><td>In vivo. Male Sprague-Dawley rats were irradiated with 56Fe particles (600 MeV/n, 12 keV/µm) at 25 cGy (25 cGy/min) or 16O particles (600 MeV/n, 189 keV/µm) at 5 cGy (5 cGy/min). NOR was performed to determine recognition learning and memory.</td><td>56Fe and 16O both resulted in an equal time spent with the novel object during the memory NOR test. The NOR test did not show significant changes in learning ability.</td></tr></table>

Time Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Hall et al., 2004</td><td>In vivo. A Swedish cohort study that analyzed military tests of learning ability, logical reasoning and spatial recognition of 2211 men 17- 18 years after β-ray, 6Ra gamma ray or X-ray radiation for cutaneous hemangioma before the age of 18 months from 1930-1959.</td><td>Irradiation at 18 months old or less resulted in significantly reduced concept discrimination and general instruction as well as technical comprehension test scores at 18 and 19 years of age.</td></tr><tr><td>Hodges et al., 1998</td><td>In vivo. Male Sprague-Dawley Rats were irradiated with X-rays at 0, 20 Gy or 25 Gy at 1.4 Gy/min. Rats were tested from 26 weeks to 44 weeks after irradiation using the T maze and water maze to assess working memory and brain damage induced by radiation.</td><td>At 29 weeks post irradiation, no learning impairments were observed in the irradiated groups during the T-maze forced alteration task. At 35 weeks post irradiation, the mean percentage of correct responses changed from 80% to 65% in both groups during the T-maze testing. At 44 weeks after irradiation, rats exposed to 25 Gy showed working memory deficits assessed by the water maze test.</td></tr><tr><td>Bellone et al., 2015</td><td>In vivo. Male B6C3F1/J mice were irradiated with 150 MeV protons at 0.5 Gy (1.5-2.5 Gy/min). A water maze was used to assess learning and memory 3 and 6 months post-irradiation.</td><td>No changes were observed in either test after 3 months. After 6 months, mice took a 1.6-fold longer distance to reach the platform.</td></tr><tr><td>Britten et al., 2012</td><td>In vivo. Wistar rats were irradiated with 125 kVp X-rays or 1 GeV/u 56Fe ions to study the effect of low dose exposure in the spatial learning ability of rats. Rats were tested for 3 days 3 months after irradiation using the Barnes maze.</td><td>3 months after exposure to 13 Gy of X-rays, rats demonstrated increased escape latency. Rats exposed to 8 and 10 Gy of X-rays experienced similar escape latency as the 0 Gy group over 3 days. Rats exposed to 56Fe ion irradiation demonstrated increased relative escape latency throughout the 3-day testing periods at all doses (0.2, 0.4 and 0.6 Gy).</td></tr><tr><td>Acharya et al., 2016</td><td>In vivo. C57Bl/6J mice were irradiated with 9 Gy of X-rays and NOR, OiP and FC were measured 5 and 6 weeks post-irradiation. The discrimination index (DI) was calculated from exploration times in familiar and novel locations. Percent of time spent freezing was used to determine FC.</td><td>5 to 6 weeks after irradiation, the DI for NOR and OiP were significantly reduced, and mice spent significantly less time frozen.</td></tr><tr><td>Forbes et al., 2014</td><td>In vivo. Male FxBN rats were irradiated with X-rays at 40 Gy from 2 fractions of 5 Gy per week over 4 weeks (each fraction at 1.25 Gy/min). Learning and memory were assessed using NOR and NOL at 3 and 18 months post-irradiation.</td><td>Irradiation significantly reduced the DR of 3-month-old rats during NOR, from 0.5 to 0.2, 3 months after irradiation. No changes in NOL were observed, and no changes at other ages and timepoints were observed.</td></tr><tr><td>Parihar et al., 2015</td><td>In vivo. Male Thy1-EGFP transgenic mice were irradiated with charged particles (16O and 48Ti) at 600 MeV/amu (0.5 to 1.0 Gy/min). NOR and OiP tests were done to assess learning and memory 6 weeks after irradiation.</td><td>After 6 weeks, impairment in cognitive ability was observed. The impairment was greater in 48Ti than 16O and greater in 30 cGy than 5 cGy.</td></tr></tbody></table>
<table><tr><td>Parihar et al., 2016</td><td>In vivo. Male Thy1-EGFP transgenic mice were irradiated with charged particles (16O and 48Ti) at 600 MeV/amu (0.05 to 0.25 Gy/min). NOR, OiP, TO and FE tests were done to assess learning and memory after 12 and 24 weeks. Wistar rats were used in the ATSET test.</td><td>Cognitive ability was lower after 24 weeks than 12 weeks in mice, but radiation was able to reduce the DI in tests at both time points.</td></tr><tr><td>Parihar et al., 2018</td><td>In vivo. Male C57BL/6 J mice were irradiated with 4He particles (400 MeV/n) at 5 cGy/min. Learning and memory were assessed using OiP and TO 6, 15 and 52 weeks after irradiation. FE was assessed 1 year after irradiation. Water maze was performed 1 year following radiation.</td><td>Learning and memory were significantly attenuated at all time points tested using all tests.</td></tr><tr><td>Impey et al., 2016</td><td>In vivo. Male C57BL/6J mice were irradiated with 600 MeV 56Fe at 0.1, 0.2 and 0.4 Gy and various dose rates from 0.25 to 0.36 Gy/min. NOR and a water maze were used to determine learning and memory ability 2 and 20 weeks post-irradiation.</td><td>Some mice at 2 weeks spent more time exploring the familiar object and less exploring the novel object. No differences were observed after 20 weeks in NOR or in the water maze test after either time point.</td></tr><tr><td>Krukowski et al., 2018b</td><td>In vivo. C57BL/6J wild type mice were exposed to alpha particles at 0, 15, 50 or 100 cGy (16.37 cGy/min). NOR was used as an assay for behavioral analysis at 90 days post-irradiation.</td><td>At late time points (90 + days post alpha exposure) mice exposed to either 15 or 50 cGy of alpha particles exhibited deficits in recognition memory as they were unable to distinguish the novel and familiar objects.</td></tr><tr><td>Krukowski et al., 2018a</td><td>In vivo. Male and female C57BL/6J wild type mice were exposed to 0, 15 or 50 cGy of GCR simulator (2.54 cGy/min). NOR assay was used to study recognition memory. A three-chamber social approach task determined social behavior and social memory.</td><td>At 45 days post-exposure, male mice exposed to 50 cGy of GCR showed significant impairments in social memory in the three-chamber social approach task compared to the 15 cGy group. NOR test revealed that radiation-induced recognition memory impairments in male cohorts only at 15 and 50 cGy.</td></tr><tr><td>Kiffer et al., 2019a</td><td>In vivo. Female C57BL/6 mice were irradiated with 16O particles (600 MeV/n) at 0.1 or 0.25 Gy (18 to 33 cGy/min). A Y-maze was performed to measure spatial memory and NOR was performed to measure non-spatial declarative memory.</td><td>After 270 days, NOR showed impaired memory. Irradiated mice at 0.1 and 0.25 Gy spent significantly more time exploring the novel object in the Y-maze, indicating no radiation-induced memory impairment.</td></tr><tr><td>Lonart et al., 2012</td><td>In vivo. Male Wistar rats were irradiated with 56Fe particles. ATSET test was used to assess simple and compound discrimination, reversal learning and set-shifting among others.</td><td>8 of 11 unirradiated rats completed all the paradigms, while only 2 of 11 irradiated rats (0.2 Gy) completed all the paradigms 90 days post-irradiation. Irradiated rats required more trial attempts to complete the simple discrimination stage compared to unirradiated rats 90 days post-irradiation.</td></tr><tr><td>Manda et al., 2007a</td><td>In vivo. Male Swiss albino mice were irradiated with 6 Gy 60Co gamma rays. Hebb-Williams maze was used to measure learning ability and spatial working memory continuously for 30 days after radiation exposure.</td><td>Time needed to reach the goal increased by 1.7-fold by day 30 after radiation, while in sham-irradiated control mice it decreased 0.2-fold in this time.</td></tr><tr><td>Rabin et al., 2012</td><td>In vivo. Male Fischer 344 rats were irradiated with various doses (50 to 100 cGy/min) of 56Fe particles (1000 MeV/n). Operant responding was used to test cognitive function. Rats were trained to press a lever to receive food at various reinforcement schedules.</td><td>Tests done 2, 4, 5, 6, 7, 8, 10 and 15 months after irradiation showed decreased cognitive function compared to controls. However, no consistent changes across time points were shown.</td></tr><tr><td>Rabin et al., 2014</td><td>In vivo. Male Sprague-Dawley rats were irradiated with various types of ionizing radiation (5 to 100 cGy/min so the time did not exceed 3 to 4 min). LET ranged from 0.22 (protons) to 181 (56Fe) keV/µm. The dose ranged from 0.1 to 200 cGy. The radiation types used were 16O (600 and 1000 MeV/n), 12C (290 MeV/n), 28Si (380, 600 and 1000 MeV/n), 48Ti (1100 MeV/n), 56Fe (600 MeV/n) and protons (1000 MeV/n). NOR was performed to assess learning and memory at various times.</td><td>16O impaired NOR performance after 2 and 4 months, but not after 10 and 12 months. 12C showed impaired NOR performance after 11 months but not 1 month. 28Si radiation showed significantly impaired NOR performance at a few doses after 4, 5, 9, 10 and 13 months, with the lowest performance occurring at 9 months. 48Ti showed impairment at 7 and 17 months. 56Fe showed impairment at 3 and 12 months. Protons showed impairment at 4 and 13 months.</td></tr><tr><td>Rabin et al., 2015</td><td>In vivo. Male Sprague-Dawley rats were irradiated with 56Fe particles (600 MeV/n, 12 keV/µm) at 25 cGy (25 cGy/min) or 16O particles (600 MeV/n, 189 keV/µm) at 5 cGy (5 cGy/min). NOR was performed within 48 h to determine recognition learning and memory.</td><td>56Fe and 16O both resulted in a 0.8-fold decrease in memory ability about 18 h after radiation. The NOR test did not show significant changes in learning ability measured about 48 h after radiation.</td></tr></table>

Known Feedforward/Feedback loops influencing this KER

TNF is a key cytokine that can initiate and promote inflammation through the activation of the NFkB pathway in glia cells. In uncontrolled conditions, TNF can lead to the development of neurodegenerative diseases as it can increase the production of pro-inflammatory cytokines and can also lead to elevated levels of iNOS, COX-2, and NOX subunits. These can then activate NADPH
oxidases to produce ROS, which can activate the NFκB pathway to amplify the overall TNF/ROS/NFκB responses to promote neuroinflammation. This process ultimately results in a feed-forward loop of chronic neurodegeneration and consequently, impaired learning and memory (Fischer and Maier, 2015).
Acharya, M. M. et al. (2016), "Elimination of microglia improves cognitive function following cranial irradiation", Scientific Reports, Vol. 6/1, Nature Publishing Group, Berlin, https://doi.org/10.1038/srep31545.

Azizova, T. V et al. (2020), "Occupational exposure to chronic ionizing radiation increases risk of Parkinson's disease incidence in Russian Mayak workers", International Journal of Epidemiology, Vol. 49/2, Oxford University Press, Oxford, https://doi.org/10.1093/ije/dyz230.

Belarbi, K. et al. (2013), "CCR2 deficiency prevents neuronal dysfunction and cognitive impairments induced by cranial irradiation", Cancer Research, Vol. 73/3, American Association for Cancer Research, California, https://doi.org/10.1158/0008-5472.CAN-12-2989.https://doi.org/10.1158/0008-5472.CAN-12-2989

Bellone, J. A. et al. (2015), "A single low dose of proton radiation induces long-term behavioral and electrophysiological changes in mice", Radiation Research, Vol. 184/2, BioOne, Washington, https://doi.org/10.1667/RR13903.1.

Britten, R. A. et al. (2012), "Low (20 cGy) doses of 1 GeV/u 56Fe-particle radiation lead to a persistent reduction in the spatial learning ability of rats", Radiation Research, Vol. 177/2, BioOne, Washington, https://doi.org/10.1667/RR2637.1.

Britten, R. A. et al. (2018), "Impaired Attentional Set-Shifting Performance after Exposure to 5 cGy of 600 MeV/n 28 Si Particles", Radiation Research, Vol. 189/3, BioOne, Washington, https://doi.org/10.1667/RR14627.1.

Cekanaviciute, E., S. Rosi and S. V. Costes. (2018), "Central nervous system responses to simulated galactic cosmic rays", International Journal of Molecular Sciences, Multidisciplinary Digital Publishing Institute (MDPI) AG, Basel, https://doi.org/10.3390/ijms19113669.

Collett, G. et al. (2020), "The psychological consequences of (perceived) ionizing radiation exposure: a review on its role in radiation-induced cognitive dysfunction", International Journal of radiation biology, Vol. 96/9, Taylor & Francis Group, London, https://doi.org/10.1080/09553002.2020.1793017.

Cucinotta, F. A. et al. (2014), "Space radiation risks to the central nervous system", Life Sciences in Space Research, Vol. 2, Elsevier Ltd, Amsterdam, https://doi.org/10.1016/j.lssr.2014.06.003.

Desai, R. I. et al. (2022), "Impact of spaceflight stressors on behavior and cognition: A molecular, neurochemical, and neurobiological perspective", Neuroscience & Biobehavioral Reviews, Vol. 138, Elsevier, Amsterdam, https://doi.org/10.1016/j.neubiorev.2022.104676.

Fischer, R. and O. Maier. (2015), "Interrelation of Oxidative Stress and Inflammation in Neurodegenerative Disease: Role of TNF", Oxidative Medicine and Cellular Longevity, Vol. 2015, Hindawi, London, https://doi.org/10.1155/2015/610813.

Forbes, M. E. et al. (2014), "Early-delayed, radiation-induced cognitive deficits in adult rats are heterogeneous and age-dependent", Radiation Research, Vol. 182/1, BioOne, Washington, https://doi.org/10.1667/RR13662.1.

Gan, H. et al. (2019), "Fluoxetine reverses brain radiation and temozolomide-induced anxiety and spatial learning and memory defect in mice", J Neurophysiol, Vol. 121, American Physiology Society, Rockville, https://doi.org/10.1152/jn.00581.2018.-Ra.

Greene-Schloesser, D. et al. (2012), "Radiation-induced brain injury: A review", Frontiers in Oncology, Vol. 2, Frontiers, Lausanne, https://doi.org/10.3389/fonc.2012.00073.

Hall, P. et al. (2004), "Effect of low doses of ionising radiation in infancy on cognitive function in adulthood: Swedish population based cohort study", British Medical Journal, Vol. 328/7430, British Medical Journal Publishing Group, London, https://doi.org/10.1136/bmj.328.7430.19.
Heisler, J. M. et al. (2015), "The Attentional Set Shifting Task: A Measure of Cognitive Flexibility in Mice", Journal of Visualized Experiments, 96, JoVe, Cambridge, https://doi.org/10.3791/51944.

Hladik, D. and S. Tapio. (2016), "Effects of ionizing radiation on the mammalian brain", Mutation Research - Reviews in Mutation Research, Vol. 770, Elsevier B.V., Amsterdam, https://doi.org/10.1016/j.mrrev.2016.08.003.

Hodges, H. et al. (1998), "Late behavioural and neuropathological effects of local brain irradiation in the rat", Behavioural Brain Research, Vol. 91/1-2, Elsevier, Amsterdam, https://doi.org/10.1016/S0166-4328(97)00108-3.

Impey, S. et al. (2016), "Short- and long-term effects of 56Fe irradiation on cognition and hippocampal DNA methylation and gene expression", BMC Genomics, Vol. 17/1, Springer Nature, Berlin, https://doi.org/10.1186/s12864-016-3110-7.

Jandial, R. et al. (2018), "Space-brain: The negative effects of space exposure on the central nervous system", Surgical Neurology International, Vol. 9/1, MedKnow Publications, Mumbai, https://doi.org/10.4103/sni.sni_250_17.

Jewell, J. S. et al. (2018), "Exposure to ≤15 cgy of 600 mev/n 56 fe particles impairs rule acquisition but not long-term memory in the attentional set-shifting assay", Radiation Research, Vol. 190/6, BioOne, Washington, https://doi.org/10.1667/RR15085.1.

Katsura, M. et al. (2021), "Recognizing Radiation-induced Changes in the Central Nervous System: Where to Look and What to Look For", RadioGraphics, Vol. 41/1, https://doi.org/10.1148/rg.2021200064.

Kiffer, F. et al. (2019a), "Late Effects of 16O-Particle Radiation on Female Social and Cognitive Behavior and Hippocampal Physiology", Radiation Research, Vol. 191/3, BioOne, Washington, https://doi.org/10.1667/RR15092.1.

Kiffer, F., M. Boerma and A. Allen. (2019b), "Behavioral effects of space radiation: A comprehensive review of animal studies", Life Sciences in Space Research, Vol. 21, Elsevier, Amsterdam, https://doi.org/10.1016/j.lssr.2019.02.004.

Krukowski, K. et al. (2018b), "Temporary microglia-depletion after cosmic radiation modifies phagocytic activity and prevents cognitive deficits", Scientific Reports, Vol. 8/1, Nature Publishing Group, Berlin, https://doi.org/10.1038/s41598-018-26039-7.

Krukowski, K. et al. (2018a), "Female mice are protected from space radiation-induced maladaptive responses", Brain, Behavior, and Immunity, Vol. 74, Academic Press Inc., Cambridge, https://doi.org/10.1016/j.bbi.2018.08.008.

Lalkovičová, M. (2022), "Neuroprotective agents effective against radiation damage of central nervous system", Neural Regeneration Research, Vol. 17/9, https://doi.org/10.4103/1673-5374.335137.

Lindsay, J. (2002), "Risk Factors for Alzheimer's Disease: A Prospective Analysis from the Canadian Study of Health and Aging", American Journal of Epidemiology, Vol. 156/5, https://doi.org/10.1093/aje/kwf074.

Lodish, H. et al. (2000), "Overview of Neuron Structure and Function", W. H. Freeman, https://www.ncbi.nlm.nih.gov/books/NBK21535/ (accessed October 27, 2021).

Lonart, G. et al. (2012), "Executive function in rats is impaired by low (20 cGy) doses of 1 GeV/u 56Fe particles", Radiation Research, Vol. 178/4, BioOne, Washington, https://doi.org/10.1667/RR2862.1.

Lopes, J. et al. (2022), "Risk of Developing Non-Cancerous Central Nervous System Diseases Due to Ionizing Radiation Exposure during Adulthood: Systematic Review and Meta-Analyses", Brain
Sciences, Vol. 12/8, Multi-Disciplinary Digital Publishing Institute (MDPI), Basel, https://doi.org/10.3390/brainsci12080984.

Manda, K. et al. (2007a), "Melatonin attenuates radiation-induced learning deficit and brain oxidative stress in mice", Acta Neurobiologiae Experimentalis, Vol. 67/1.

Manda, K. et al. (2007b), "Radiation-induced cognitive dysfunction and cerebellar oxidative stress in mice: Protective effect of α-lipoic acid", Behavioural Brain Research, Vol. 177/1, Elsevier, Amsterdam, https://doi.org/10.1016/j.bbr.2006.11.013.

Mhatre, S. D. et al. (2022), "Neuro-consequences of the spaceflight environment", Neuroscience & Biobehavioral Reviews, Vol. 132, Elsevier, Amsterdam, https://doi.org/10.1016/j.neubiorev.2021.09.055.

Miry, O. et al. (2021), "Life-long brain compensatory responses to galactic cosmic radiation exposure", Scientific Reports 2021 11:1, Vol. 11/1, Nature Publishing Group, https://doi.org/10.1038/s41598-021-83447-y.

Mousa, A. and M. Bakhiet. (2013), "Role of Cytokine Signaling during Nervous System Development", International Journal of Molecular Sciences, Vol. 14/7, Multidisciplinary digital Publishing Institute (MDPI), Basel, https://doi.org/10.3390/ijms140713931.

National Council on Radiation Protection and Measures (NCRP). (2016). Commentary No. 25 – Potential for central nervous system effects from radiation exposure during space activities phase I: Overview.

Parihar, V. K. et al. (2016), "Cosmic radiation exposure and persistent cognitive dysfunction", Scientific Reports, Vol. 6/1, Nature Publishing Group, Berlin, https://doi.org/10.1038/srep34774.

Parihar, V. K. et al. (2015), "What happens to your brain on the way to Mars", Science Advances, Vol. 1/4, American Association for the Advancement of Science, Washington, https://doi.org/10.1126/sciadv.1400256.

Parihar, V. K. et al. (2018), "Persistent nature of alterations in cognition and neuronal circuit excitability after exposure to simulated cosmic radiation in mice", Experimental Neurology, Vol. 305, Academic Press Inc., Cambridge, https://doi.org/10.1016/j.expneurol.2018.03.009.

Pariset, E. et al. (2021), "Ionizing radiation-induced risks to the central nervous system and countermeasures in cellular and rodent models", International Journal of Radiation Biology, Vol. 97/sup1, Taylor & Francis Group, London, https://doi.org/10.1080/09553002.2020.1820598.

Pasqual, E. et al. (2021), "Cognitive effects of low dose of ionizing radiation – Lessons learned and research gaps from epidemiological and biological studies", Environment International, Vol. 147, Elsevier, Amsterdam, https://doi.org/10.1016/j.envint.2020.106295.

Prieto, G. A. and C. W. Cotman. (2017), "Cytokines and cytokine networks target neurons to modulate long-term potentiation", Cytokine & Growth Factor Reviews, Vol. 34, Elsevier, Amsterdam, https://doi.org/10.1016/j.cytogfr.2017.03.005.

Rabin, B. M. et al. (2012), "Interaction between age of irradiation and age of testing in the disruption of operant performance using a ground-based model for exposure to cosmic rays", Age, Vol. 34/1, Springer Nature, Berlin, https://doi.org/10.1007/s11357-011-9226-4.

Rabin, B. M. et al. (2015), "Acute effects of exposure to 56Fe and 16O particles on learning and memory", Radiation Research, Vol. 184/2, BioOne, Washington, https://doi.org/10.1667/RR13935.1.

Rabin, B. M., B. Shukitt-Hale and K. L. Carrihill-Knoll. (2014), "Effects of Age on the Disruption of Cognitive Performance by Exposure to Space Radiation", Journal of Behavioral and Brain Science, Vol. 04/07, Scientific Research Publishing, Inc, Wuhan, https://doi.org/10.4236/jbbs.2014.47031.
Turnquist, C., B. T. Harris and C. C. Harris. (2020), "Radiation-induced brain injury: current concepts and therapeutic strategies targeting neuroinflammation", Neuro-Oncology Advances, Vol. 2/1, Oxford University Press, Oxford, https://doi.org/10.1093/noajnl/vdaa057.
## AOPs Referencing Relationship

<table><thead><tr><td>AOP Name</td><td>Adjacency</td><td>Weight of Evidence</td><td>Quantitative Understanding</td></tr></thead><tbody><tr><td>AOP: 483 - Deposition of Energy Leading to Learning and Memory Impairment</td><td>non-adjacent</td><td>Moderate</td><td>Low</td></tr></tbody></table>

## Evidence Supporting Applicability of this Relationship

Evidence for this relationship comes from human, rat, and mouse models, with most of the evidence in mice. The relationship is not sex or life stage specific.

### Taxonomic Applicability

<table><thead><tr><td>Term</td><td>Scientific Term</td><td>Evidence</td><td>Link</td></tr></thead><tbody><tr><td>human</td><td>Homo sapiens</td><td>Low</td><td>NCBI</td></tr><tr><td>mouse</td><td>Mus musculus</td><td>Moderate</td><td>NCBI</td></tr><tr><td>rat</td><td>Rattus norvegicus</td><td>Low</td><td>NCBI</td></tr></tbody></table>

### Life Stage Applicability

<table><thead><tr><td>Term</td><td>Evidence</td></tr></thead><tbody><tr><td>All life stages</td><td>Moderate</td></tr></tbody></table>

### Sex Applicability

<table><thead><tr><td>Sex</td><td>Evidence</td></tr></thead><tbody><tr><td>Unspecific</td><td>Moderate</td></tr></tbody></table>

## Key Event Relationship Description

Inflammatory mediators such as IL-1β, TNF-α, and IL-6, can influence the normal behavior of neuronal cells and their functional connection. Overexpression of pro-inflammatory mediators disrupts the integrity and function of the neuronal network through decreased neurogenesis, synaptic complexity and increased necrosis and demyelination, ultimately impairing learning and memory (Cekanaviciute, Rosi, & Costes, 2018; Fan & Pang, 2017). Impaired short-term and long-term memory, as well as associative learning are consequences of the dysregulated expression of pro-inflammatory cytokines as reported in behavioral paradigms (Donzis & Tronson, 2014).

Under physiological conditions, cytokine levels are low but greatly increased in response to various insults. Cytokines mediate immune response through ligand binding to cell surface receptors, which activate signaling cascades such as the JAK-STAT or MAPK pathways to produce or recruit more cytokines. Once organs initiate inflammatory reactions, the cytokines can modulate different metabolic and molecular pathways that have direct effects on neurons or indirect effects mediated by microglia, astrocytes or vascular endothelial cells (Mousa & Bakhiet, 2013; Prieto & Cotman, 2018). Modulation of these pathways ultimately affects crucial neuronal networks such as that within the hippocampus, which is one of the main brain regions responsible for learning and memory (Barrientos et al., 2015; Bourgognon & Cavanagh, 2020).

## Evidence Supporting this KER

The strategy for collating the evidence to support the relationship is described in Kozbenko et al 2022. Briefly, a scoping review methodology was used to prioritize studies based on a population, exposure, outcome, endpoint statement.

Overall Weight of Evidence: Moderate

## Biological Plausibility
Several reviews provide support of biological plausibility between increase in pro-inflammatory mediators and impaired learning and memory. In the central nervous system, cytokines and their receptors are constitutively expressed and affect brain plasticity, which is the ability to modify its activity and connections in response to intrinsic or extrinsic stimuli. In a pathological state, pro-inflammatory, or Th-1 type cytokines become particularly relevant in the brain and these cytokines bind to their receptors to induce a conformational change and activate intracellular signaling pathways (Mousa & Bakhiet, 2013). The main cytokines presenting detrimental effects are IL-1β, TNF-α and IL-6, as these are the most studied.

The mechanism by which these cytokines modify learning and memory processes are not clearly understood due to the complexity of inflammatory signaling, although it involves alterations in the neural circuits that regulate these processes (Bourgognon & Cavanagh, 2020; Pugh et al., 2001). Multiple studies have demonstrated that IL-1β presents a critical role in the formation of hippocampal dependent memory, as IL-1β and its receptor are highly expressed in the hippocampus. Experimentally elevated levels of IL-1β in the hippocampus lead to impaired performance in behavioral paradigms such as spatial memory, contextual learning, and passive avoidance tasks (Donzis & Tronson, 2014; Pugh et al. 2001; Yirmiya & Goshen, 2011).

There are several possible mechanisms for this detrimental effect. One proposed mechanism is through reduced N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor functions, both of which are involved in long-term potentiation, a process that strengthens synaptic connections between neurons. Other potential mechanisms for the effects of IL-1β on brain plasticity and memory can involve the activation of several pathways such as p38 mitogen-activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK), caspase 1, nuclear factor kappa B (NF-κB), and brain-derived neurotrophic factor (BDNF). These complex pathways have roles in neuronal health, long-term potentiation, brain plasticity and ultimately learning and memory (Patterson, 2015). The mechanisms of IL-6 and TNF-α in terms of impaired cognition also remain unclear, although the pathways that these cytokines activate are similar to those of IL-1β due to the network of cytokine interactions (Donzis & Tronson, 2014).

## Empirical Evidence

The empirical evidence collected for this KER comes from in-vivo studies and various methods used to assess the impairment in learning and memory. The behavioral paradigms test associative learning, as well as long and short-term memory (Bourgognon & Cavanagh, 2020).

Various studies also examined the presence of pro-inflammatory mediators induced by lipopolysaccharide (LPS) injections (Sparkman et al., 2006), Escherichia coli (E. coli) injections (Barrientos et al., 2009), direct cytokine injections (Gonzalez et al., 2009; Goshen et al., 2007; Taepavarapruk & Song, 2010), ionizing radiation (Bhat et al., 2020; Jenrow et al., 2013), surgical procedures (Tan et al., 2014) and transgenic models with overexpressed pro-inflammatory cytokines (Hein et al., 2010; Heyser et al., 1997; Moore et al., 2009).

## Dose Concordance

Dose concordance relating an increase in pro-inflammatory mediators leading to an impaired learning and memory was demonstrated using various stressors, including ionizing radiation, LPS, IL-1β, and E. coli injections.

A 10 Gy X-ray irradiation in mice led to an increase in IL-6 secretion compared to dimethyl sulfoxide (DMSO) control. Impaired learning and memory were assessed by a decrease in freezing time in fear conditioning tests and a decrease in novel object recognition and object in place discrimination index (DI) (Bhat et al., 2020). A 10 Gy gamma irradiation of rats' brains similarly indicated impaired cognitive function and an increase in OX-6+ cell density, indicative of inflammation (Jenrow et al., 2013).

Rats injected with 2.5x10⁹ CFU of E. coli showed a significant increase in IL-1β in multiple regions of the brain, spleen and serum, along with impaired memory and learning as indicated by fear
conditioning (Barrientos et al., 2009). Mice injected with 100 µg of LPS demonstrated an increase in IL-1β, TNF-α, IL-6 and IL-10 levels, while the performance in a Morris water maze was impaired, indicating impairments in spatial memory (Sparkman et al., 2006).

## Time Concordance

Various studies show that an increase in pro-inflammatory mediators is observed before or at the same time as impaired learning and memory. Some studies observe each event at the same time at specific timepoints. In mice injected with LPS, an increase in IL-1β, TNF-α, IL-6 and IL-10 was observed after 4 hours, while impaired learning and memory were also consistently observed 4 h after LPS injection (Sparkman et al., 2006). Both key events were also found in rats from 4 h to 8 days after E. coli injection (Barrientos et al., 2009). Cohort studies using age as a stressor in humans found increased pro-inflammatory mediators and decreased cognitive function over 6 months (Holmes et al., 2009) and 7 years (Alley et al., 2008). Many studies observe increased pro-inflammatory mediators occurring before impaired learning and memory. Rats with hippocampal injections of IL-1β showed impaired memory 24 h and 7 days later (Gonzalez et al., 2009). Rats showed increased hippocampal IL-1β and IL-6 levels 6 h after surgery to expose the right carotid artery, while learning and memory was impaired 2 weeks later (Tan et al., 2014). When mice were irradiated with X-rays, IL-6 was found to increase 24 h later, while learning and memory was impaired 5 weeks later (Bhat et al., 2020). Rats irradiated with gamma rays showed increased inflammation after 2 months, while memory was impaired after 6 months (Jenrow et al., 2013).

## Incidence Concordance

Some evidence also shows that pro-inflammatory mediators increase equal to or greater than the amount that impairs learning and memory at the same stressor severity. Many of these studies were done with transgenic mice expressing IL-1β or IL6 as a stressor and showed increased levels of various pro-inflammatory mediators including ICAM-1, CCL2, IL-1α, COX-1 and MCP-1 from 2- to 147-fold, while learning and memory were decreased a maximum of 0.6-fold (Hein et al., 2010; Heyser et al., 1997; Moore et al., 2009).

## Other Evidence

Multiple longitudinal cohort studies followed hundreds to thousands of older adults over the course of several years ranging from four to sixteen years and examined the relationship between pro-inflammatory marker levels and the rate of cognitive change over time. These studies reported linear negative correlations between IL-6 and TNF-α levels and learning and memory ability as the populations aged (Alley et al., 2010; Holmes et al., 2009; Schram et al., 2007).

## Essentiality

Studies show that overexpression of pro-inflammatory cytokines affect learning and memory, and several treatments that alter the effects/function of pro-inflammatory mediators preserve cognitive function. The treatments included MW-151, a selective inhibitor of pro-inflammatory cytokine production, lidocaine, an anesthetic with anti-inflammatory properties, ethyl-eicosapentaenoate (E-EPA) and 1-[(4-nitrophenyl)sulfonyl]-4-phenylpiperazine (NSPP), both of which are anti-inflammatory drugs and α-melanocyte stimulating hormone (α-MSH), which antagonizes the effects of pro-inflammatory cytokines through G protein coupled receptors (Bhat et al., 2020; Gonzalez et al., 2009; Jenrow et al., 2013; Taepavarapruk and Song, 2010; Tan et al., 2014). More details are provided in the Modulating Factors section below.

## Uncertainties and Inconsistencies

* Due to the indirect linkage between the two key events, there is no clear understanding of how increases in pro-inflammatory mediators cause impaired learning and memory. (Donzis & Tronson, 2014).

* A previous prospective population-based cohort study found an association between antihistamine use and increased risk of dementia, which is the loss of cognitive functioning. Therefore, anti-
inflammatory medications such as antihistamines could modulate the progression to impaired learning and memory (Gray et al., 2015).

## Known modulating factors

<table><thead><tr><td>Modulating factor</td><td>Details</td><td>Effects on the KER</td><td>References</td></tr></thead><tbody><tr><td>Drug</td><td>NSPP (anti-inflammatory drug)</td><td>There was a 0.35-fold decrease in IL-6 levels compared to controls when 10 Gy irradiated mice were treated with NSPP. Mice that were exposed to whole brain irradiation (10 Gy) and treated with NSPP (5 mg/kg) exhibited significantly improved performance in novel object recognition and object in place.</td><td>Bhat et al., 2020</td></tr><tr><td>Drug</td><td>α-MSH (modulator of action of pro-inflammatory cytokines)</td><td>α-MSH injected into the hippocampus prevented the IL-1β-induced decrease in contextual fear memory.</td><td>Gonzalez et al., 2009</td></tr><tr><td>Drug</td><td>MW-151 (inhibitor of pro-inflammatory microglial cytokine production)</td><td>Treatment decreased the OX-6+ cell density and restored memory.</td><td>Jenrow et al., 2013</td></tr><tr><td>Drug</td><td>Lidocaine (an anti-inflammatory local anesthetic)</td><td>Lidocaine treatment restored IL-6 levels and improved memory.</td><td>Tan et al., 2014</td></tr><tr><td>Age</td><td>Young age</td><td>Rats aged 3 months showed increased IL-1β levels in the hippocampus for less time than in mice aged 24 months. Hippocampal-dependent memory was impaired in the old mice. The inflammatory response is shorter and less severe in young individuals, leading to reduced cognitive impairment.</td><td>Barrientos et al., 2009; Barrientos et al., 2012</td></tr><tr><td>Drug</td><td>E-EPA (known to improve cognitive function through reducing inflammation)</td><td>E-EPA reduced the increase in IL-6 expression and improved memory to control levels.</td><td>Taepavarapruk & Song, 2010</td></tr></tbody></table>

## Quantitative Understanding of the Linkage

The table below provides some representative examples of quantitative linkages between the two key events. It was difficult to identify a general trend across all the studies due to differences in experimental design and reporting of the data. All data is statistically significant unless otherwise stated.

### Dose Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Sparkman et al., 2006</td><td>In-vivo. Three-month-old male C57BL/6 mice were injected with 100 µg of LPS. Pro-inflammatory mediator levels were determined with immunohistochemical staining. Spatial working memory was evaluated through a Morris water maze.</td><td>IL-1β increased from 15 pg/mL (control) to 400 pg/mL. TNF-α increased from &lt;5 pg/mL (control) to 360 pg/mL. IL-6 increased from undetectable levels (control) to 300 pg/mL. IL-10 increased from undetectable levels (control) to 260 pg/mL. Performance in the water maze was impaired in distance swam, latency and swim speed after LPS injection, with a maximum 2-fold increased swim distance.</td></tr><tr><td>Taepavarapruk & Song, 2010</td><td>In-vivo. Male Long-Evans rats were administered 15 ng/µL/day of IL-1β for either 1 or 7 days. IL-1 expression was determined, and memory was assessed with an eight-arm radial maze.</td><td>IL-1β administration of 105 ng/µL resulted in a 2.5-fold increase in IL-1 expression and a 1.5-fold increase in number of entries, indicating memory impairment.</td></tr><tr><td>Goshen et al., 2007</td><td>In-vivo. 2-4-month-old male mice were injected with IL-1β and IL-1ra (IL-1 receptor antagonist) at 1 or 10 ng (injected in 10 µL) doses. IL-1β was assessed by quantitative real time RT-PCR, and hippocampal IL-1ra was determined by ELISA. Contextual fear conditioning was used to assess associative learning and memory. Evaluations were taken after 3 weeks of recovery. Water maze was used for spatial memory testing.</td><td>Mice that were injected with a high dose of IL-1β (10 ng) had shorter freezing times, indicating impaired contextual fear conditioning, whereas mice injected with a low dose of IL-1β (1 ng) showed longer freezing times, indicating improved contextual fear conditioning. Spatial memory was impaired in IL-1raTG rats (astrocyte-directed overexpression of IL-1ra) as latency and path length was increased (non-significantly) in the majority of the trials compared to wild type rats.</td></tr><tr><td>Gonzalez et al., 2009</td><td>In-vivo. Adult male Wistar rats had hippocampal injections of 5 ng/0.25 µL of IL-1β post-conditioning and memory was assessed through freezing behavior.</td><td>Rats injected with 5 ng/0.25 µL IL-1β displayed impaired contextual fear memory, where injected rats spent 0.6-fold less time freezing.</td></tr></tbody></table>
<table><tr><td>Bhat et al., 2020</td><td>In-vivo. Female mice were exposed to X-ray irradiation at 0, 2, 4 and 10 Gy (5.519 Gy/min). Novel object recognition, object in place and fear conditioning evaluated memory. IL-6 levels were measured by ELISA.</td><td>There was a significant 2.97-fold increase in the secretion of IL-6 in cells irradiated with 10 Gy compared to DMSO control. DMSO was used as a control as treatment mice were also treated with NSPP solubilized in DMSO. Irradiated mice demonstrated a significant decrease in DI for novel object recognition and object in place. Irradiated mice also spent significantly less time freezing in context fear, context gen and pre-tone assessments of fear conditioning.</td></tr><tr><td>Jenrow et al., 2013</td><td>In-vivo. Adult male Fischer 344 rats' brains were irradiated with 10 Gy gamma rays. OX-6 levels (indicative of inflammation levels) were determined, and novel object recognition was performed to assess memory.</td><td>10 Gy increased OX-6+ cell density from 1493±270 to 1966±218 cells/mm3. Also, after 10 Gy, the discrimination ratio for novel object recognition decreased from 68.76±11.30% to 21.71±10.86%.</td></tr><tr><td>Barrientos et al., 2009</td><td>In-vivo. Male F344xBN F1 rats, either old (24 months) or young (3 months), received an injection of 2.5x10<sup>9</sup> CFU of E. coli. IL-1β levels were determined using ELISA and fear conditioning was performed to assess learning and memory.</td><td>Significant increases in IL-1β were observed in the hippocampus, hypothalamus, parietal cortex, serum and spleen after injection, with a maximum 4-fold increase. Injected mice also spent 0.6-fold less time freezing.</td></tr></table>

Time Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Sparkman et al., 2006</td><td>In-vivo. Three-month-old male C57BL/6 mice were injected with 100 µg of LPS. Pro-inflammatory mediator levels were determined with immunohistochemical staining and measured 4 h after LPS injection. Spatial working memory was evaluated 4 h after LPS injection through a Morris water maze.</td><td>IL-1β increased from 15 pg/mL (control) to 400 pg/mL. TNF-α increased from &lt;5 pg/mL (control) to 360 pg/mL. IL-6 increased from undetectable levels (control) to 300 pg/mL. IL-10 increased from undetectable levels (control) to 260 pg/mL. Performance in the water maze was found impaired in distance swam, latency and swim speed 4 h after LPS injection, with a maximum 2-fold increased swim distance.</td></tr><tr><td>Gonzalez et al., 2009</td><td>In-vivo. Adult male Wistar rats had hippocampal injections of 5 ng/0.25 µL of IL-1β post-conditioning and memory was assessed through freezing behavior.</td><td>Rats spent 0.6- to 0.7-fold less time freezing 24 h after injection with IL-1β. Rats also showed impaired long-term memory 7 days after injection when they spent 0.7-fold less time frozen.</td></tr><tr><td>Bhat et al., 2020</td><td>In-vivo. Female mice were exposed to X-ray irradiation at 0, 2, 4 and 10 Gy (5.519 Gy/min). Novel object recognition, object in place and fear conditioning evaluated memory. IL-6 levels were measured by ELISA.</td><td>24 hours after exposure to 10 Gy irradiation, mice showed a significant 2.97-fold increase in the secretion of IL-6 compared to DMSO control (DMSO was used as a control as mice were also treated with NSPP solubilized in DMSO). At week 5, irradiated mice demonstrated a significant decrease in DI for novel object recognition and object in place, and spent less time freezing in context fear, context gen and pre-tone assessments of fear conditioning.</td></tr><tr><td>Jenrow et al., 2013</td><td>Adult male Fischer 344 rats' brains were irradiated with 10 Gy 137Cs gamma rays. OX-6 levels (indicative of inflammation levels) were determined, and novel object recognition was performed to assess memory.</td><td>After 2 months, OX-6+ cell density increased from 1493±270) to 1966±218 cells/mm3. A similar but smaller increase was found after 9 months. Only measured after 6 months, the discrimination ratio for novel object recognition decreased from 68.76±11.30% to 21.71±10.86%.</td></tr><tr><td>Tan et al., 2014</td><td>In-vivo. Four-month-old male Fischer 344 rats had 1 cm of right carotid artery dissected free from surrounding tissue in a 15-min surgery. IL-6 and IL-1β levels were determined by western blot and a Barnes maze was used to test spatial learning and memory.</td><td>Surgery increased IL-1β and IL-6 in the hippocampus by 2- to 3-fold compared to controls 6 h after surgery. Additionally, rats in the surgery group took 2- to 3-fold more time to identify the target in the Barnes maze task 2 weeks after surgery.</td></tr><tr><td>Barrientos et al., 2009</td><td>In-vivo. Male F344xBN F1 rats, either old (24 months) or young (3 months), received an injection of 2.5x10<sup>9</sup> CFU of E. coli. IL-1β levels were determined using ELISA and fear conditioning was performed to assess hippocampal-dependent learning and memory.</td><td>Significant increases in IL-1β were observed in the hippocampus, hypothalamus, parietal cortex, serum and spleen most frequently 4 h after injection, with a maximum 4-fold increase. IL-1β in the hypothalamus was also increased in old mice up to 8 days after injection. Old mice spent less time freezing 4 days after injection, while old injected mice also spent 0.6-fold less time freezing 8 days after injection.</td></tr><tr><td>Alley et al., 2008</td><td>In-vivo. A cohort study of older adults aged 70-79 years that were tested in 1988, 1991 and 1995 to determine changes in cognitive functioning and IL-6 levels. ELISA was used to measure IL-6 levels. Cognitive function was determined by various tests, including spatial recognition, spatial ability, verbal recall, language and abstraction. Short Portable</td><td>As IL-6 levels increased, mean cognitive scores decreased compared to baseline cognitive scores. A linear inverse association was found between inflammation and general cognitive scores, both measured at the end of the 7-year study.</td></tr></tbody></table>
<table><tr><td></td><td>Mental Status Questionnaire (SPMSQ) was used as a measure of cognitive performance. Participants were re-interviewed at 2.5 and 7 years.</td><td></td></tr><tr><td>Holmes et al., 2009</td><td>In-vivo. A cohort study of subjects with mild to severe Alzheimer's disease who were cognitively assessed and tested for inflammatory markers. Cognitive assessments were performed using the Alzheimer's Disease Assessment Scale (ADAS-COG) test. The sandwich immunoassay multiplex cytokine assay measured TNF-α at 2, 4 and 6 months</td><td>Subjects with high TNF-α levels (3.2 [standard error (SE) 0.6]) at baseline observed greater changes in ADAS-COG over 6-months compared to subjects with low TNF-α (0.8 [SE 0.8]). The mean change in ADAS-COG score was 2.6 (±7.0) points over the 6 months.</td></tr></table>

Incidence Concordance

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Moore et al., 2009</td><td>In-vivo. Transgenic mice overexpressing IL-1β (activated by microinjection of FIV-Cre) for 2 weeks underwent spatial and non-spatial behavioral tasks using a Morris water maze. IL-1β, IL-1α and MCP-1 were measured by RT-PCR.</td><td>Measurement of IL-1β in hippocampal tissue revealed mRNA levels increased 22.9-fold. The pro-inflammatory mediators IL-1α and MCP-1 mRNA levels were also increased 3.1- and 147-fold, respectively. Overexpression of IL-1β in the hippocampus hindered acquisition and long-term memory retention on the spatial task but did not impact non-spatial learning.</td></tr><tr><td>Hein et al., 2010</td><td>In-vivo. Male and female IL-1βXAT mice on a C57BL/6 background (containing a dormant human IL-1β gene activated by a virus expressing Cre) were injected with 1.5x10<sup>4</sup> viral particles of the feline immunodeficiency virus (expresses Cre) in the hippocampus. Fear conditioning and a Morris water maze were performed to assess learning and memory.</td><td>IL-1β was increased 15-fold in the hippocampus, and the expression of other pro-inflammatory mediators CCL2, IL-1α and COX-1 were similarly increased. Mice spent 0.6-fold less time freezing and 0.8-fold less time in the target quadrant, indicating impaired learning and memory. No differences between males and females were observed.</td></tr><tr><td>Heyser et al., 1997</td><td>In-vivo. C57BL/6 x SJL hybrid mice with a GFAP-IL6 fusion gene were tested for avoidance learning and expression of ICAM-1.</td><td>Compared to non-transgenic (+/+) mice, ICAM-1 was increased 2-fold at 3 months old and 4-fold at 12 months old in both heterozygous (+/tg) and homozygous (tg/tg) transgenic mice. Also compared to +/- mice, +/-tg mice showed impaired avoidance response at 12 months old, where tg/tg mice showed impaired avoidance at 3, 6 and 12 months old.</td></tr></tbody></table>

Other Evidence

<table><thead><tr><td>Reference</td><td>Experiment Description</td><td>Result</td></tr></thead><tbody><tr><td>Alley et al., 2008</td><td>In-vivo. A cohort study of older adults aged 70 to 79 years that were tested in 1988, 1991 and 1995 to determine changes in cognitive functioning and IL-6 levels. ELISA was used to measure IL-6 levels. Cognitive function was determined by various tests, including spatial recognition, spatial ability, verbal recall, language and abstraction. Short Portable Mental Status Questionnaire (SPMSQ) was used as a measure of cognitive performance. Participants were re-interviewed at 2.5 and 7 years.</td><td>As IL-6 levels increased, mean cognitive scores decreased compared to baseline cognitive scores. A linear inverse association was found between inflammation and general cognitive scores, both measured over the course of 7 years. Participants in the top IL-6 tertile (IL-6 &gt; 3.8 pg/mL) had 62% increased odds of declines in global cognitive function (Odds Ratio (OR) = 1.62, 95% Confidence Interval (CI), 1.07–2.45). They also had 88% increased odds of cognitive impairment, (OR = 1.88, 95% CI, 1.20–2.94), relative to those with lower levels of IL-6.</td></tr><tr><td>Holmes et al., 2009</td><td>In-vivo. A cohort study of subjects with mild to severe Alzheimer's disease were cognitively assessed and tested for inflammatory markers. Cognitive assessments were performed using the Alzheimer's Disease Assessment Scale (ADAS-COG) test. The sandwich immunoassay multiplex cytokine assay measured TNF-α at 2, 4 and 6 months</td><td>Subjects with high TNF-α levels (3.2 [SE 0.6]) at baseline observed greater changes in ADAS-COG over 6-months compared to subjects with low TNF-α (0.8 [SE 0.8]). The mean change in ADAS-COG score was 2.6 (SD 7.0) points over the 6 months.</td></tr><tr><td>Schram et al., 2007</td><td>In-vivo. The Leiden 85-plus Study, performed with participants aged 85 to 90 years (n= 705), assessed memory function and its association with inflammatory markers. IL-6 plasma levels were measured by ELISA. C-reactive protein (CRP) pro-inflammatory mediator was measured by Rate Near Infrared Particle Immunoassay. The 12-Picture Learning Test was used to evaluate memory function.</td><td>When levels of pro-inflammatory mediators were higher than baseline, delayed recall memory point estimate was negative, indicating impaired memory.</td></tr></tbody></table>
Alley, D. E. et al. (2008), "Inflammation and Rate of Cognitive Change in High-Functioning Older Adults", The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, Vol. 63/1, Oxford University Press, Oxford, https://doi.org/10.1093/gerona/63.1.50.

Alley, D. E. et al. (2008), "Inflammation and Rate of Cognitive Change in High-Functioning Older Adults", The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, Vol. 63/1, https://doi.org/10.1093/gerona/63.1.50.

Barrientos, R. M. et al. (2015), "Neuroinflammation in the normal aging hippocampus", Neuroscience, Vol. 309, Elsevier, Amsterdam, https://doi.org/10.1016/j.neuroscience.2015.03.007.

Barrientos, R. M. et al. (2009), "Time course of hippocampal IL-1 β and memory consolidation impairments in aging rats following peripheral infection", Brain, Behavior, and Immunity, Vol. 23/1, Elsevier, Amsterdam, https://doi.org/10.1016/j.bbi.2008.07.002.

Barrientos, R. M. et al. (2012), "Aging-related changes in neuroimmune-endocrine function: Implications for hippocampal-dependent cognition", Hormones and Behavior, Vol. 62/3, Elsevier, Amsterdam, https://doi.org/10.1016/j.yhbeh.2012.02.010.

Bhat, K. et al. (2020), "1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine treatment after brain irradiation preserves cognitive function in mice", Neuro-Oncology, Vol. 22/10, Oxford University Press, Oxford, https://doi.org/10.1093/neuonc/noaa095.

Bourgognon, J.-M. and J. Cavanagh. (2020), "The role of cytokines in modulating learning and memory and brain plasticity", Brain and Neuroscience Advances, Vol. 4, https://doi.org/10.1177/2398212820979802.

Cekanaviciute, E., S. Rosi and S. Costes. (2018), "Central Nervous System Responses to Simulated Galactic Cosmic Rays", International Journal of Molecular Sciences, Vol. 19/11, MDPI, Basel, https://doi.org/10.3390/ijms19113669.

Donzis, E. J. and N. C. Tronson. (2014), "Modulation of learning and memory by cytokines: Signaling mechanisms and long term consequences", Neurobiology of Learning and Memory, Vol. 115, Elsevier, Amsterdam, https://doi.org/10.1016/j.nlm.2014.08.008.

Fan, L. W. and Y. Pang. (2017), "Dysregulation of neurogenesis by neuroinflammation: key differences in neurodevelopmental and neurological disorders", Neural Regeneration Research, Vol. 12/3, Wolters Kluwer, https://doi.org/10.4103/1673-5374.202926.

Gonzalez, P. V. et al. (2009), "Memory impairment induced by IL-1β is reversed by α-MSH through central melanocortin-4 receptors", Brain, Behavior, and Immunity, Vol. 23/6, Elsevier, Amsterdam, https://doi.org/10.1016/j.bbi.2009.03.001.

Goshen, I. et al. (2007), "A dual role for interleukin-1 in hippocampal-dependent memory processes", Psychoneuroendocrinology, Vol. 32/8-10, Elsevier, Amsterdam, https://doi.org/10.1016/j.psyneuen.2007.09.004.

Gray, S. L. et al. (2015), "Cumulative Use of Strong Anticholinergics and Incident Dementia", JAMA Internal Medicine, Vol. 175/3, https://doi.org/10.1001/jamainternmed.2014.7663.
Hein, A. M. et al. (2010), "Sustained hippocampal IL-1β overexpression impairs contextual and spatial memory in transgenic mice", Brain, Behavior, and Immunity, Vol. 24/2, Elsevier, Amsterdam, https://doi.org/10.1016/j.bbi.2009.10.002.

Heyser, C. J. et al. (1997), "Progressive decline in avoidance learning paralleled by inflammatory neurodegeneration in transgenic mice expressing interleukin 6 in the brain", Proceedings of the National Academy of Sciences, Vol. 94/4, National Academy of Sciences, Washington, https://doi.org/10.1073/pnas.94.4.1500.

Holmes, C. et al. (2009), "Systemic inflammation and disease progression in Alzheimer disease", Neurology, Vol. 73/10, Wolters Kluwer, https://doi.org/10.1212/WNL.0b013e3181b6bb95.

Jenrow, K. A. et al. (2013), "Selective Inhibition of Microglia-Mediated Neuroinflammation Mitigates Radiation-Induced Cognitive Impairment", Radiation Research, Vol. 179/5, BioOne, https://doi.org/10.1667/RR3026.1.

Moore, A. H. et al. (2009), "Sustained expression of interleukin-1β in mouse hippocampus impairs spatial memory", Neuroscience, Vol. 164/4, Elsevier, Amsterdam, https://doi.org/10.1016/j.neuroscience.2009.08.073.

Mousa, A. and M. Bakhiet. (2013), "Role of Cytokine Signaling during Nervous System Development", International Journal of Molecular Sciences, Vol. 14/7, MDPI, Basel, https://doi.org/10.3390/ijms140713931.

Patterson, S. L. (2015), "Immune dysregulation and cognitive vulnerability in the aging brain: Interactions of microglia, IL-1β, BDNF and synaptic plasticity", Neuropharmacology, Vol. 96, Elsevier, Amsterdam, https://doi.org/10.1016/j.neuropharm.2014.12.020.

Prieto, G. A. and C. W. Cotman. (2017), "Cytokines and cytokine networks target neurons to modulate long-term potentiation", Cytokine & Growth Factor Reviews, Vol. 34, Elsevier, Amsterdam, https://doi.org/10.1016/j.cytogfr.2017.03.005.

Pugh, C. R. et al. (2001), "The immune system and memory consolidation: a role for the cytokine IL-1β", Neuroscience & Biobehavioral Reviews, Vol. 25/1, Elsevier, Amsterdam, https://doi.org/10.1016/S0149-7634(00)00048-8.

Schram, M. T. et al. (2007), "Systemic Markers of Inflammation and Cognitive Decline in Old Age", Journal of the American Geriatrics Society, Vol. 55/5, Wiley, https://doi.org/10.1111/j.1532-5415.2007.01159.x.

Sparkman, N. L. et al. (2006), "Interleukin-6 Facilitates Lipopolysaccharide-Induced Disruption in Working Memory and Expression of Other Proinflammatory Cytokines in Hippocampal Neuronal Cell Layers", Journal of Neuroscience, Vol. 26/42, Society for Neuroscience, https://doi.org/10.1523/JNEUROSCI.3376-06.2006.

Taepavarapruk, P. and C. Song. (2010), "Reductions of acetylcholine release and nerve growth factor expression are correlated with memory impairment induced by interleukin-1β administrations: effects of omega-3 fatty acid EPA treatment", Journal of Neurochemistry, Vol. 112/4, Wiley https://doi.org/10.1111/j.1471-4159.2009.06524.x.

Tan, H. et al. (2014), "Critical role of inflammatory cytokines in impairing biochemical processes for learning and memory after surgery in rats", Journal of Neuroinflammation, Vol. 11/1, Springer Nature, https://doi.org/10.1186/1742-2094-11-93.
Yirmiya, R. and I. Goshen. (2011), "Immune modulation of learning, memory, neural plasticity and neurogenesis", Brain, Behavior, and Immunity, Vol. 25/2, Elsevier, Amsterdam, https://doi.org/10.1016/j.bbi.2010.10.015.