Early and long-term alterations in sensorimotor parameters following fractionated gamma irradiation in an experimental setting

INTRODUCTION

Increased interest in radiation neurobiology is largely due to research in space medicine and the need to ensure protection against space radiation during future manned interplanetary flights. The salient characteristics of space radiation, comprised of Earth’s radiation belts (protons and electrons), solar cosmic rays (protons), and galactic cosmic rays (accelerated hydrogen and helium ions, heavy charged particles), lead to significant non-uniformity of the radiation dose both in space and time due to the unpredictability of solar proton events. Therefore, research into the effects of chronic and protracted fractionated irradiation, including the study of mechanisms underlying central nervous system impairments, is of particular importance for space radiobiology [1]. In addition to the use of radiation therapy for cancer patients and the widespread application of medical diagnostic procedures involving radiation exposure, a comprehensive analysis of the impact of protracted, chronic, or fractionated irradiation on the brain is driven by the need to protect the health of nuclear industry professionals.

For a number of radiation-induced effects on the central nervous system, a causal relationship with radiation exposure has been established including the determination of threshold values for absorbed dose. For instance, acute exposure exceeding 1–2 Gy in adults or 0.1 Gy in young children may increase the risk of cognitive dysfunction (International Commission on Radiological Protection Publication 118³). Cognitive dysfunction in rodents, which is manifested as impaired memory or reduced learning ability in humans, along with impaired spatial learning, novel object recognition, and decision-making function, is associated with radiation effects, primarily in the hippocampal region [2]. The mechanisms underlying these impairments involve radiation-induced oxidative stress, which disrupts normal proliferation and differentiation of neuronal stem cells, impairs synaptic plasticity, triggers the activation of pro-inflammatory cytokines, and, at high doses, leads to neuronal death [2].

Regarding other consequences of ionizing radiation exposure on the brain, there is less epidemiological and experimental data. For instance, several studies discuss the potential increased risk of developing neurodegenerative diseases long after radiation exposure [3]. In a cohort of Russian nuclear industry workers chronically exposed to radiation during their professional activities at a nuclear fuel production facility (Mayak Production Association, Ozersk, Chelyabinsk Region), an analysis of the dose-response relationship, accounting for non-radiation factors (sex, age), revealed an association between the incidence of Parkinson’s disease and the cumulative dose from external gamma radiation [4].

In a study of causes of death in a French cohort of nuclear industry workers (SELTINE, 1968–2014), statistically significant dose-risk relationships were established for increased mortality from leukemia and dementia, although the authors noted that the observed association between the risk of death from dementia and low-dose radiation should be interpreted with caution at this stage [5]. However, for scenarios of acute exposure in the dose range of 0–4 Gy (Japanese cohort of atomic bomb survivors in Hiroshima and Nagasaki), studies did not find an increased incidence of dementia [6] or a correlation between age-related cognitive decline and radiation dose [7].

In a study carried out on patients with multiple sclerosis in the period prior to the onset of symptoms related to this disease and confirmation of the diagnosis, Motamed et al. found that this cohort had been more frequently exposed to diagnostic or therapeutic X-ray irradiation and had a higher total accumulated dose than individuals in the control group without multiple sclerosis; based on this, the authors concluded a possible association between the dose of X-ray irradiation and the risk of developing this disease [8].

In a study investigating the risks of developing non-cancer central nervous system diseases following exposure in adulthood, Lopes et al. revealed a significant positive relative risk for the development of cerebrovascular diseases and Parkinson’s disease; however, the authors noted several methodological issues, such as difficulties in reconstructing individual radiation doses and limited accounting for potential confounding factors [9]. Particular challenges when studying the mechanisms of ionizing radiation’s influence on neurodegenerative and age-related changes arise as a result of the long latency period from exposure to the potential development of disease signs.

Currently, there is a lack of longitudinal studies to establish causal relationships between radiation dose and potential risks of developing neurodegenerative diseases [2][3].

At the same time, no attempts have been made to study the association between the development of neurodegenerative diseases long after irradiation in experimental animals [3]. On the one hand, radiation-induced pro-inflammatory microglial activation and oxidative stress increase the risk of neurodegenerative processes; on the other hand, certain irradiation regimens have demonstrated a neuroprotective effect in experiments carried out on a transgenic animal model of Alzheimer’s disease [10].

One of the simplest and most effective methods for the experimental assessment of neurodegenerative disease symptoms using laboratory animals involves investigations into motor function [11]. Sensorimotor effects measured in animals following experimental exposures indirectly indicate alterations in other behavioral parameters, such as cognitive functions and emotions [12]. A number of behavioral tests developed to assess sensorimotor functions in rodents enable the evaluation of sensorimotor information processing speed, locomotor function, balance, grip strength, motor coordination, orienting-exploratory response, etc. [12].

The present study investigates early and long-term alterations in sensorimotor parameters characterizing coordination in laboratory animals subjected to fractionated gamma irradiation at a young age across various doses. The work, which is a continuation of previously published research into early alterations in sensorimotor parameters in fractionally irradiated mice [13], aims to supplement the previously obtained results with information on the first identified impairment of coordination in irradiated animals in the long term after exposure.

MATERIALS AND METHODS

The experiment was performed on mice of both sexes (n = 400) of the C57Bl/6 strain (Specific Pathogen Free (SPF), vivarium of the Institute of Cytology and Genetics, SB RAS, Novosibirsk). The animals were subjected to fractionated gamma-irradiation from birth until 30 days. Subsequent assessments of sensorimotor parameters were conducted in the irradiated animals four times over a period of 18 months. Five observation groups were formed: two control groups and three groups with different radiation levels, each group being comprised of 80 individuals each (40 males and 40 females). With age, the number of examined animals gradually decreased due to the natural death of some of them.

The exposed animal groups included the “5 Gy” group (n = 80), the “1 Gy” group (n = 80), and the “0.1 Gy” group (n = 80). Mice in these groups were exposed to cumulative doses of 5 Gy, 1 Gy, and 0.1 Gy, respectively. In each case, the cumulative dose was divided into 20 equal fractions of 0.25 Gy, 0.05 Gy, and 0.005 Gy, respectively. Irradiation was performed over four weeks, five days per week, using the experimental radiobiological setup IGR-1M (Kvant, Russia), equipped with ¹³⁷Cs sources. For exposure to single doses of 0.25 Gy and 0.05 Gy, the dose rate was 0.72 Gy/min. To achieve a single dose of 0.005 Gy, lead collimators were used (the dose rate when using collimators was 10 mGy/min). The non-uniformity of the gamma field within the working space did not exceed 10%. Mice were exposed in their permanent housing cages; lactating females were temporarily removed during the exposure procedure.

The control groups of non-exposed mice included: a group of intact animals comprising the biological control (BC) (n = 80) and a sham-exposed placebo group (0 Gy) (n = 80). The animals in the “0 Gy” group underwent the same manipulations as those in the exposed groups (with the exception of the radiation exposure itself) to account for the influence of stress factors associated with animal handling and temporary maternal separation on the studied parameters.

Following the completion of the exposure, the animals at an age of 28–30 days were transferred to separate cages (10 males and 10 females per cage) for their permanent accommodation in a conventional vivarium (at a temperature of 22 ± 2°C, with unlimited access to drinking water and complete granulated feed).

To assess sensorimotor function, the animals were tested at the ages of 1, 6, 12, and 18 months.

The raised-beam walking test, designed to assess motor deficits primarily in the hind limbs, can reveal pathologies of the motor cortex and evaluate coordination in rodents [14][15]. The test evaluated the ability of an individual animal to traverse a 1-meter-long, elevated, tapering, two-level beam. Over two days, the mice were trained to traverse the beam to reach a safe shelter. On the third day, the traversal of the setup by the mice was recorded on video (Sony α37 camera).

The following parameters were recorded: total time taken to traverse the beam from the start point to the shelter, foot slips off the beam surface, falls, and the number of limb placements on the lower ledge (errors) [14][15]. Parameters were recorded using the RealTimer software (Open Science RPC, Russia). If an animal fell off the setup or failed to reach the shelter during testing on the 3^rd day, it was excluded from further analysis (in each experimental group, the number of such animals did not exceed 1–2 individuals).

The results were expressed as mean values and standard errors (M ± SE). Since all analyzed parameters conformed to a normal distribution according to the Kolmogorov–Smirnov test, comparison of mean values between experimental and control groups was performed using parametric methods with Student’s t-test. Results were considered statistically significant at α = 0.05.

Pearson’s correlation analysis was performed to assess the association of the studied parameters characterizing sensorimotor function in the animals. Regression analysis was used to describe the influence of age on the investigated parameters. Multifactorial analysis of variance (ANOVA) in the general linear model was used to identify the effects of radiation dose and other concomitant factors (sex, age, radiation-associated stress). When assessing the stress factor, it was assumed that only the intact animals in the BC group did not experience stress associated with the early-life exposure procedure. Data were analyzed using Microsoft Excel and the R programming language.⁴ Pairwise comparisons of parameter values across different dose groups were performed to establish the significance of differences between groups exposed to different doses. For this purpose, data were normalized for age and sex, followed by post-hoc analysis in a one-way ANOVA with Bonferroni correction.

RESULTS AND DISCUSSION

During the raised-beam walking test, the time taken by the animal to traverse a 1-m distance on an elevated, tapering beam was recorded, along with events such as limb placements on the lower ledge of the setup (errors), limb slips off the beam, and pauses.

Table 1 presents the measured parameters of the raised-beam walking test for mice in the different experimental groups. Generally, a lower number of errors, pauses, and/or a shorter beam traversal time was observed in females compared to males within the same group. For instance, statistically significant differences between males and females were noted at the age of 1 month in the “0 Gy” and “1 Gy” groups for the parameter of total walking time, as well as in the “0.1 Gy” group for the number of errors.

At 6 months of age, females exhibited a statistically significant reduction compared to males in the number of pauses in the BC group, the number of errors in the “0.1 Gy” group, and the walking time in the “1 Gy” group. At 12 months, a statistically significant reduction in total walking time in females was observed in the BC, “0.1 Gy”, and “1 Gy” groups; a reduction in the number of pauses was seen in the “0.1 Gy” and “1 Gy” groups; a reduction in the number of slips occurred in the BC and “5 Gy” groups; and a reduction in the number of errors was found in the “0 Gy” and “0.1 Gy” groups. At 18 months, a reduction in total walking time was noted in the BC, “0 Gy”, and “1 Gy” groups, while a reduction in the number of pauses was observed in the BC, “0 Gy”, and “0.1 Gy” groups (Table 1).

From the comparison of sensorimotor function parameters in non-exposed mice from the “0 Gy” and BC groups, no definitive conclusions can be drawn regarding the influence of the stress factor on these parameters. For instance, at 1 month, females in the “0 Gy” group made fewer errors than females in the BC group, whereas at 6 months, the number of errors in “0 Gy” females became statistically significantly higher than in the BC group. At 12 months, animals of both sexes in the “0 Gy” group had a shorter total walking time compared to the BC group; additionally, males made fewer slips and errors relative to the BC group, which could be interpreted as improved coordination. At 18 months, males in the “0 Gy” group had statistically significantly fewer errors than the BC group, but a longer total walking time (Table 1).

Age-related coordination impairment is one of the signs of altered sensorimotor functions accompanying neurodegenerative diseases. A decline in coordination observed as the animals aged was manifested by an increase in beam traversal time and an increased number of pauses. In non-exposed animals (BC and “0 Gy” groups), regression analysis indicated that the relationship between age and the analyzed parameters could be best described by a linear function. A statistically significant effect of age on total walking time (R² = 0.06; F = 29.5; p << 0.001) was manifested by an average increase in walking time of 0.112 ± 0.021 seconds per month. A statistically significant positive linear relationship was also found between age and the number of pauses during beam traversal (R² = 0.03; F = 12.6; p < 0.001), along with a negative relationship between age and the number of slips (R² = 0.02; F = 7.6; p = 0.006). Regression analysis did not reveal a statistically significant relationship between age and the number of errors (F = 1.3; p = 0.26).

Multifactorial analysis of variance using the general linear model to assess the influence of the studied factors on sensorimotor function parameters showed that factors such as age, radiation dose, and sex had a statistically significant influence on all investigated sensorimotor parameters (Table 2). In contrast, a statistically significant influence of the stress factor was noted only for the number of limb slips (F = 7.5; p = 0.006). Based on the results of the multifactorial ANOVA, the time required to traverse the beam increased with age and radiation dose more markedly in males than in females. However, the dependence on radiation dose was non-linear: the minimum traversal times were characteristic of animals exposed to a dose of 0.1 Gy, while in those exposed to 5 Gy, this parameter reached values comparable to the non-exposed control. Similar patterns were observed for the number of pauses during the test. The number of errors during beam traversal increased linearly with both the age of the mice and the radiation dose, being maximal at a dose of 5 Gy and more pronounced in males than in females. The number of slips decreased with age but increased with radiation dose to a greater extent in males than in females.

To assess the effect of radiation exposure on sensorimotor function parameters in mice, the previously identified sex and age differences were taken into account. For this purpose, the studied parameters were standardized by sex and age: after setting the values for non-exposed animals to 1, the measurements for exposed males and females were normalized to the mean value of the corresponding parameter for males or females in the control group of the same age. Furthermore, since the multifactorial analysis did not reveal a substantial influence of the stress factor on the studied parameters despite comparisons between the “0 Gy” and “BC” groups using Student’s t-test showing non-systematic differences across ages, the exposed animals were compared to a combined control group (BC + “0 Gy”) to describe the changes in their sensorimotor parameters.

The dependence of the investigated parameters on the dose of fractionated radiation for animals of different ages is presented in Figures 1–4.

The study demonstrates the effect of fractionated gamma radiation dose on sensorimotor function. However, the dependence of the investigated parameters on the dose was non-linear. A long-lasting effect (persisting at least until 18 months of age in mice) of improved sensorimotor parameters (coordination) was identified in animals irradiated with a dose of 0.1 Gy compared to non-exposed animals. Specifically, at any age, the time required to traverse the beam and the number of pauses in the “0.1 Gy” group were statistically significantly lower than in the non-exposed animals (Figs. 1, 2). The total walking time decreased by 15–30%, and the number of pauses decreased by 25–40%. At the age of 1 month, the number of errors in this group was also statistically significantly reduced (Fig. 3), measuring 7.6 ± 0.33 in the control group versus 6.2 ± 0.50 in the “0.1 Gy” group (t = 2.28; p = 0.023).

At 12 months of age, a reduction in the number of slips (Fig. 4) to 0.4 ± 0.11 was observed, compared to 0.70 ± 0.071 in the control group (t = 2.46; p = 0.01). The decrease in these parameters can be interpreted as a long-term improvement in coordination in animals exposed to a dose of 0.1 Gy.

In the “1 Gy” group, the walking time was statistically significantly shorter by 10–15% as compared to the non-exposed control at the ages of 1, 6, and 18 months (Fig. 1), the number of pauses was lower by 20% at 1 and 18 months (Fig. 2), while the number of slips was reduced by 10% at 1 month and by 40% at 12 months (Fig. 4). On the other hand, the number of errors made by animals in the “1 Gy” group during the test exceeded the control values at 6 months: 10.4 ± 0.52 errors compared to 8.5 ± 0.39 errors in the control group (t = 3.03; p = 0.003). At 18 months: 10.1 ± 0.54 errors compared to 7.9 ± 0.43 errors in the control group (t = 3.36; p < 0.001). The corresponding data are presented in Figure 3.

When the radiation dose was increased to 5 Gy, the total walking time and the number of errors and slips increased; however, they did not exceed control values until the age of 12 months. In animals irradiated with a dose of 5 Gy, the beam traversal time was statistically significantly reduced compared to the control at 6 and 18 months (Fig. 1), while at 12 months, an increase in the mean walking time to 6.3 ± 0.2 s was observed compared to 5.7 ± 0.1 s in the control group (t = 2.46; p = 0.014). The number of pauses in this group was lower than in the control only in animals at 1 month of age: 2.3 ± 0.22 pauses compared to 3.0 ± 0.20 pauses in the control (t = 2.39; p = 0.018) (Fig. 2).

Conversely, in the long term after exposure to a dose of 5 Gy, the number of errors in performing the test was statistically significantly increased in mice at 12 months: 10.6 ± 0.63 errors versus 8.7 ± 0.37 errors in the control (t = 2.68; p = 0.008). At 18 months: 12.7 ± 0.61 errors versus 7.9 ± 0.43 errors in the control (t = 6.52; p < 0.001) (Fig. 3). At 18 months, the number of limb slips from the beam also increased to 0.72 ± 0.13 as compared to a control value of 0.36 ± 0.05 (t = 3.05; p = 0.002) (Fig. 4). These changes indicate a deterioration in sensorimotor parameters in animals during the long-term period following the completion of fractionated irradiation.

Thus, at the final examination time point (18 months of age), signs of diminished coordination capacity were observed in animals irradiated with a cumulative dose of 5 Gy: although the total walking time did not differ from that of non-exposed animals, mice in the “5 Gy” group made a statistically significant greater number of errors and exhibited more limb slips from the beam. These alterations can be interpreted as indicative of earlier age-related decline in sensorimotor function compared to the control group.

Pairwise comparisons of parameters normalized for sex and age across different dose groups were performed using post-hoc analysis via a one-way ANOVA with Bonferroni correction. It was found that for the criteria of walking time and number of pauses, the “0.1 Gy” group was statistically significantly different from all other experimental groups (p << 0.001), while the “1 Gy” and “5 Gy” groups showed no significant differences from each other in these parameters. Regarding the number of errors, the “0.1 Gy” group was statistically significantly different from the “1 Gy” and “5 Gy” groups (p < 0.001), but did not differ from the non-exposed animals. Conversely, there were no significant differences in this parameter between the “5 Gy” and “1 Gy” groups. The standardized number of slips in the “0.1 Gy” group was lower than in the “5 Gy” group (p = 0.039).

Correlation analysis revealed the presence of a moderate direct correlation between various parameters of the raised-beam walking test. Specifically, a moderate direct correlation was demonstrated between walking time and the number of pauses (r = 0.67; p < 0.001); a correlation was also identified between age and total walking time (r = 0.32; p < 0.001). The obtained data verify the suitability of the employed tool for assessing coordination in animals. When coordination is impaired, the animal grips the beam tightly apparently fearing a fall, which prolongs the walking time. Conversely, the better the animal’s coordination, the shorter the total walking time, and the fewer errors, slips, and pauses it makes.

The investigations of sensorimotor function in mice exposed to radiation during early development discussed in this paper constitute part of a larger experiment assessing various parameters of higher nervous activity and brain status following fractionated irradiation. For instance, at the age of 2 months, we identified enhanced cognitive function (spatial learning) in the Barnes maze in animals exposed to a cumulative dose of 0.1 Gy [16]. Furthermore, over the course of their lifespan, these same mice exhibited more anxious, neophobic behavior in a marble burying test [17]. Immunohistochemical analysis of the hippocampal dentate gyrus in mice exposed to 0.1 Gy revealed significant stimulation of neurogenesis (elevated levels of PROM1⁺ cells), as well as an increase in the proportion and number of GAP43⁺ cells expressing neuromodulin, a protein associated with synaptogenesis [16]. It is plausible that the observed alterations in neurogenesis and synaptic plasticity, along with the stimulation of spatial learning, more pronounced neophobic behavior, and increased anxiety in animals exposed to 0.1 Gy, may also be related to the described increase in the speed of movement along the elevated, narrowing beam.

Some studies have reported impaired animal coordination in the inclined beam walking test immediately following fractionated irradiation (5 fractions of 0.1 Gy of X-ray irradiation daily, cumulative dose 0.5 Gy) [18], which was also accompanied by increased anxiety and locomotor activity in the open field test. Our study did not detect early coordination impairments following irradiation at cumulative doses of 0.1–5 Gy. However, it is important to consider the different, more protracted fractionation regimen with the lower doses per fraction used in our work.

Over a longer-term period, other authors have described alterations in sensorimotor function detected by the acoustic startle response test: following single irradiation at 10 weeks of age with a dose of 0.5 Gy, animals exhibited a decreased response speed to an acoustic stimulus; this effect persisted until 12 months of age; at 12 and 18 months, these animals showed reduced locomotor activity in the open field test [19]. Conversely, while irradiation at a low dose (0.063 Gy) did not induce early effects, at 18 months of age, an increase in exploratory activity in the open field and an improved acoustic startle response compared to control animals were demonstrated. These data are consistent with the effects of low (0.1 Gy) and high (5 Gy) dose irradiation observed in our study.

Overall, the neuroprotective effect of low-dose irradiation based on criteria such as reduced pro-inflammatory microglial activation, enhanced cognitive function, and stimulated neurogenesis has been demonstrated in a number of studies [10]. Regarding high-dose irradiation, the primary mechanisms underlying cognitive decline are considered to be the disruption of adult neurogenesis in the neurogenic niches of the brain (in the hippocampus and cerebellum), pro-inflammatory microglial activation, and impairment of redox status, energy metabolism, and mitochondrial function [2]. Alongside cognitive changes, these same effects could potentially also lead to impaired coordination. For instance, although this particular study did not assess motor function in the irradiated animals, there is evidence of reduced neurogenic activity and the development of neuroinflammation in the cerebellum of rats following fractionated high-dose irradiation [20]. As a continuation of our analysis, it would be advisable to conduct histological examinations of the cerebellum and motor cortex in animals subjected to fractionated irradiation at cumulative doses of 0.1 Gy and 5 Gy during the long-term period post-exposure, to identify morphological correlates of the observed behavioral changes.

CONCLUSION

The study investigated alterations in sensorimotor parameters characterizing coordination in laboratory animals subjected to fractionated gamma radiation at different doses during early development. Fractionated gamma irradiation induces multidirectional, dose-dependent changes in sensorimotor function in mice, which are observed both in the short-term and long-term periods. Immediately following the completion of fractionated irradiation, a stimulatory effect of exposure at a cumulative dose of 0.1 Gy on sensorimotor parameters in mice was demonstrated. This observed improvement in coordination following 0.1 Gy exposure persisted up to the age of 18 months. In the case of irradiation at a cumulative dose of 5 Gy, signs of impaired motor coordination in mice were identified for the first time in this experiment during the long-term period post-exposure, whereas no signs of sensorimotor function disruption were detected within the first 6 months after irradiation.

Authors’ contributions. All authors confirm that their authorship complies with the ICMJE criteria. The author contributions are distributed as follows: Natalya I. Atamanyuk — manuscript drafting; Nadezhda A. Obvintseva — experiments conducting, data collection; Irina A. Shaposhnikova — statistical analysis of data; Sergey S. Andreev — development of methodologies and procedures; Evgeny A. Pryakhin — scientific supervision of the project.