Proton-induced Behavioral Alterations approved Cognitive Dysfunction

Proton-induced Behavioral Alterations approved Cognitive Dysfunction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Proton-induced Behavioral Alterations approved Cognitive Dysfunction Gyutae Kim, Kyu-Sung Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3893200/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A long-term space exploration faces extremely hazardous environmental stresses, and the exposure to space radiation has been considered as one of the most dangerous factors in Space. For the safety of astronauts, even a low amount of proton exposure in space radiation is necessary to be understood to protect their body systems and maintain the functional balance. However, the endpoint test based on behavioral responses lead to an undetermined consequence in the functional declines by producing the inconsistent results despite of the accumulated proofs in the molecular and the cellular damages by proton. To clarify the systemic link between the proton exposure and the cognitive decline in behaviors, some fundamental behavioral tests, such as rota-rod, open field test, and novel object recognition, were revisited, and the proton-induced alteration was examined by computing behavioral markers in time basis. A portion out of total population (28 SD rats) involved for each test, and the relevant behavioral markers in time were calculated to assess the proton-induced effects in emotion, locomotion and memory. The examination in 3 months of behavioral responses after the different amount of exposure (control, 30cGy-, and 1Gy-exposed animals) identified the emotional and locomotive alterations while few memory-related changes were observed. The computed behavioral markers suggested a quantitative approach to demonstrate the cognitive behavioral effects by a low amount of proton (30cGy) as well as the direct relation between the proton exposure and the cognitive alteration. Cognition Behavioral Response Space Environment Radiation Hypergravity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Current NASA’s projects have issued 5 hazardous factors, such as space radiation, gravity alteration, closed environments, isolation, and distance from Earth during space exploration. These potential risks induce various structural and functional abnormalities in molecules, cells, and neurons as well as organ systems [ 1 ]. Of the addressed factors, space radiation is one of the most influential risks to damage a wide range of biological levels from DNA [ 2 , 3 ] to central nervous system [ 4 – 6 ]. Space radiation is composed of various types of energies and linear energy transfer (LTE), and most destructive effects were known to be generated by the heavy ionic particles (1–2%) while proton is a dominant type (up to 91%) [ 7 , 8 ]. Due to the damage degrees, many previous studies have focused on the physiological and functional alterations by the heavy ionic particles, underestimating those by proton. At the early stage of space exploration, the amount of radiation exposure was limited, but its impacts have increased as space mission expanded to Mars (expected dose: 350mSv/year for 3 years) [ 9 , 10 ]. In general, the heavy ions, such as hydrogen ( 1 H 1 ), carbon ( 12 C 6 ), silicone ( 28 S 14 ), titanium ( 48 Ti 22 ), and iron ( 56 Fe 26 ), damage DNA by producing lesion-specific enzymes to break its double-strand [ 2 ]. Once the series of damages are initiated, the radiation-induced effects sequentially expand to the damages at upper biological levels, like chromosomal aberrations [ 11 ], cell kinetics [ 12 ], mitotic death [ 13 ], peripheral blood lymphocytes [ 14 ]. Furthermore, the triggered damages affect the functions of central nervous system as well as inner organs [ 5 , 15 – 17 ]. As shown in these previous studies, various biological targets have been examined to understand the radiation-induced effects, and the relevant interest is moving to the function of cognition as the staying duration increased in Space [ 18 – 20 ]. Proton, which is the dominant type in space radiation as mentioned, has been mainly used for the medical purpose, specifically cancer therapy [ 21 – 23 ]. The repeated treatment by proton exposure raised the concerns of cognitive impairment, and some studies demonstrated the relation between the proton exposure and the cognition-related functional decline [ 21 , 22 , 24 – 26 ]. However, the cognition-related behavioral alteration as the functional endpoint after proton exposure is still ambiguous while the neurochemical [ 27 , 28 ], neurophysiological [ 25 ] and functional images [ 22 , 29 ] have indicated the proton-induced damages in the brain. Thus, many studies emphasized various proton-induced molecular and neuronal damages with no direct equivocal behavioral characteristics. Especially, the biological consequence under a low amount of proton (< 1Gy) has been rarely investigated while some heavy ions induced a loss of bone mass by the same amount [ 30 ]. Here, we proposed some behavioral markers, which were computed based on the moving distance, the mean speed, and the resting time in open field test, to assess the behavioral alteration after the proton exposure. In addition, the behavioral tests, such as novel object recognition and rota-rod tests, were also adopted to estimate the functional changes of cognition, and their own markers were generated to examine their superiority to those in open field test. Based on computed behavioral markers, we identified the cognition-related changes in open field test, and the suggested analysis quantitatively distinguished the behavioral responses to a low amount of proton (30cGy) from normal activities. Considering the characteristics of the radiation-induced effects, which gradually spread over the biological systems during a long time, the relevant impacts needed to be analyzed in respect to time, and our current analysis adopted the hypothesis in the time basis. This helped to generalize the proton-induced behavioral effects, and it also demonstrated the time relation between the cognitive behavioral responses and the amount of radiation exposure. Methods All procedures and principles of laboratory animal care were approved by the Animal Ethics Committee at Inha University (INHA 220822-835). Total 28 rats (Sprague Dawley, M) were used in this study, and the animals were divided into 3 groups (control, 30cGy-, and 1Gy-exposed animals) based on the amount of proton exposure. Out of total population, some selective animals were used for the assessment of the changes in weight, rota-rod test, open field test, and novel object recognition at scheduled periods for behavioral markers (Fig. 1 ). All animals were housed in the controlled environments, such as stable temperature (22–25℃), humidity (40–60%), ventilation (10–15 times/hour), static pressure difference (> 5mmAq), noise level (< 60dB) and a 12:12 hours light-dark cycle except during the experiments. Animal preparation & Proton Exposure Before the relocation for the proton exposure, some selected animals (n = 21) were initially trained to attain a prerequisite level (180–200 sec) of the motional balance on the rota-rod test. Once the animals’ balance level was ready, the animals were relocated for the proton beam exposure at Korea Multi-purpose Accelerator Complex (KOMAC, KyungJu, South Korea). The animals were moved by a ground transportation (approximately 350 km) one day before the proton exposure for the animals’ stability. After 24-hours stabilization, an animal was anesthetized by an intramuscular injection of a mixed solution of Ketamine (1 µl/g) and Xylazine (0.33 µl/g), and it was placed in the self-produced holding frame. The frame with an animal was positioned at about 1.5 m away from the beam outlet. Using a 100-MeV beam energy, single proton exposure was conducted. The effective area of exposure was 10×10 cm 2 , and its uniformity was 95.93%. The beam exposure was completed by multiple beam pulses (energy per pulse: 0.0026–0.0035 Gy/pulse), and two final doses were 30 cGy and 1 Gy (error rate < 2.5%). Initially, the weight change and skin damage were estimated the stresses of the beam exposure and the long transportation, and these changes were again inspected at the sacrificing period (72 hours, 4 weeks, and 3 months) for a molecular examination. The weight change of animals was examined based on the comparison with the normal weight curve (Hilltop Lab Animals, Inc., Pennsylvania, US). On the normal weight chart, all animals’ weights were overlapped to identify the effects of the long-distance transportation and radiation exposure. The normality of animals’ condition was confirmed by the weights. Especially, any weights below the normal one at a given time were counted as the result by the long transportation or the proton exposure. Considering that the radiation-related symptoms generally appeared in 1–2 weeks later since exposure, the early change in the weights was assumed as the consequence by the long-term transportation for the experiment of proton exposure. Rota-Rod test Rota-rod (RR) test estimated coordinated motional balance after the proton exposure. Some selected animals (n = 18) walked on the rolling rod with a constant speed (34 meter/min), and one RR test lasted for approximately 3 minutes, which repeated in 5 times with a five-minute break. The test was ceased if the animal changed its walking direction oppositely or the animal was just hanging the rolling rod with no walk. The test was generally conducted at 3, 7, and 10 days before the exposure, and at 3, 7, 28, 42, 56, 70, 84, 86, and 88 days after the proton exposure. Before the relocation for the proton exposure, all animals were trained to reach a required walking level, which was ranged between 180 and 200 seconds, and the applied regulations for the test were the same before and after the exposure. The divided groups based on the amount of proton exposure performed the RR test on the scheduled days, and the attained time on the rod was used to assess the motional balance. Once the animal reached the required time for the test, the RR test was ceased. The overall results of RR test from the different animal groups were separately plotted in a closed form containing all the measurements to predict their changing patterns of test performance. Using a custom-written code (MATLAB, MathWorks, US), all the measurements in time (days) were presented up to 3 months, and a smoothed area based on the measurements was calculated. The shape of the computed area showed the predicted pattern in each animal group. Open Field test Using 14 out of total animals, open field (OPF) test was designed for the quantitative evaluation of animal’s locomotor activity as well as the animals’ moving pattern. OPF test was performed at 7, 14, 21, 28, 35, 56, 77, and 84 days after the proton exposure. Specific parameters in OPF test were measured, such as total distance, resting time, and mean speed with or without resting, using a video tracking system (SMART 3.0, Harvard/PANLab, US). During a 10-minute (600 seconds) test, animals’ movements were detected and calculated in the defined area, so-called the zone of a background (1 x 1 m 2 ), and the total distance was measured based on the automated detection. Distance in zone showed the total moving length in the given area. The relevant results were presented by their means and standard deviation (STD) (m) at the planned periods. A linear regression was applied on the means of distances in time to assess the proton effects. The resting (immobile) time was defined by a certain speed (< 0.5 cm/sec) which indicated the animal showed little movement during the detection. Once the total distance and resting time were confirmed, the mean speed with or without resting time was calculated as follows; $${Mean Speed}_{without resting}=\frac{\text{T}\text{o}\text{t}\text{a}\text{l} \text{D}\text{i}\text{s}\text{t}\text{a}\text{n}\text{c}\text{e}}{600-\text{r}\text{e}\text{s}\text{t}\text{i}\text{n}\text{g} \text{t}\text{i}\text{m}\text{e}}$$ 1 $${Mean Speed}_{with resting}=\frac{\text{T}\text{o}\text{t}\text{a}\text{l} \text{D}\text{i}\text{s}\text{t}\text{a}\text{n}\text{c}\text{e}}{600}$$ 2 Combining with total moving distance, the mean speed (cm/sec) was presented in bar charts with or without the resting time. Fear/Anxiety-like Behavior Test Fifteen animals out of the total population were used to assess the levels of fear/anxiety-like behavior. An animal freely moved in the same area of open field (1 x 1 m 2 ), and all movements were recorded. Using the recorded movie (10 frames / sec), the duration (sec) of in- or out-of-center was measured, and the time duration in each specified area was mainly used to estimate the level of fear. Each animal’s location in either in-center or out-of-center was determined by an imaginary square (50 x 50 cm 2 ), positioned at the center of the open field. Thus, the condition of in-center implied that the animal located in the imaginary square, and that of out-of-center did that the location of animal was out of the square. At the selected periods (1,3,4,5,8,11, and 12 weeks after proton exposure), the durations of in- and out-of-center were measured, and the individual (open circle) and the averaged time (± standard deviation) (closed circle) were presented. Using the averages at the periods, the overall means (dotted lines) of in- and out-of-center were computed to show the level of fear/anxiety-like behavior by the different amount of proton exposure. Novel Object Recognition Novel object recognition (NOR) test assessed an animal’s ability to identify a newly presented object, which the cognitive function was involved with. The test initially began by placing an animal in a squared area (1 x 1 m 2 ) with two same objects (aluminium-material, 245ml in volume, 9 x 6.5 cm 2 in height x diameter). For a given time (10 minutes), the animal was exposed to these objects, and it became eventually accustomed to them, so called familiar objects (FO) (the first searching session). These objects were located at randomized two corners in the squared area, and the animal was allowed to freely search the zones, physically contacting two FOs. The identification of an object was defined as the time which the animal was located at around the objects (generally, the defined zone within 4 cm away from the surface of the object). Once the adapting time was completed for the familiarity to FOs, the animal took a rest (60 minutes). After the break, one of FOs was replaced by a new object (glass-material, 155ml in volume, 14 x 3.5 cm 2 in height x diameter), and the animal again underwent the second searching session (10 minutes). Some selected animals (n = 18) were used for this test. As explained, the animal’s cognitive function was measured by the staying time in the defined zone. As the new object was recognized by the animal, the animal was expected to stay in the zone with the new object longer than that with FO. For a separate analysis, 3 zones were defined; the zone with FO as old zone, that with new object as new zone, and the background with no object. The main behavioral markers in NOR test were the number of entries to zone and the staying time in zone, which estimated the recognition of a newly presented object. Statistical Analysis For the statistical evaluation of results, 2-sampled t-test and 2-way analysis of variance (ANOVA) were adopted for the analysis of weights and NOR test, respectively. The weights were tested if the results of two proton-exposed groups were significantly different from that of control group, and the same test was applied to identify any significance at multiple periods of time (less than 1st, 4th, and 12th week). The results of NOR was examined based on the number of entry and staying time with a given time intervals (1st, 3rd, 4th, 5th, 8th, 11th, and 12th week). At these periods of time after the proton exposure, the number of entry and staying time in the defined zones were calculated based on the relative percentages. Using these computed values, the statistical analysis by ANOVA was performed. The level of significance was 0.01, and all statistical analysis were conducted by a user-written code in MATLAB (MathWorks, US). Statistical comparison in fear/anxiety-like behavior test was conducted by t-test (significance: 0.01). For each group (control, 30cGy- & 100cGy-exposed groups), 5 animals’ locations in or out of center were measured at the given period (1st, 3rd, 4th, 5th, 8th, 11th, and 12th week), and these individual durations at the periods were compared to show how much the exposed proton affected the function of fear/anxiety-like behavior by lapse of time. Results Animals were trained to be familiar with the rota-rod (RR) test, and they repeated the test until they reached the required level of performance before the transportation for the proton exposure (see Rota- Rod test ). Once the animals were ready for the RR test, they were relocated to the laboratory in Korea Atomic Energy Research Institute (KOMAC, KyungJu, South Korea) one-day before the proton experiment. After the exposure was completed, the animals were transported back to Inha lab. The change of weights indicated the effects of the long transportation and the proton exposure were minor (Fig. 2 ). Comparing with the referenced weights in days (band in gray), no group showed any significant difference in the weights (p > 0.059, 2-sampled t-test), except that between the control and 100 cGy-exposed group at 12th week (p = 0.042, 2-sampled t-test). Thus, the result indicated no early effect by the proton exposure as well as by the long transportation, but the amount of proton, 100 cGy, generated a proton-induced effect in a long-term period (> 12 weeks). The overall changes in animals’ weights up to 12 weeks also supported this inference, and the weights of all groups remained in the expected range of the referenced weights (Fig. 2 A). On the other hand, the regressions using the weights at three periods (3, 28, and 84 days after the exposure) anticipated an early effect by the long transportation and the proton exposure, but the increased difference between the control and proton-exposed groups indicated the effect was mainly caused by the proton exposure instead by the long transportation which was limited at the early period (Fig. 2 B). Therefore, the difference of weights between the control and the proton-exposed groups was resulted by the proton exposure while that by the long transportation was limited. All animals attained the expected level of the performance in RR test within a week, and the relevant capability was generally maintained even after the proton exposure (Fig. 3 ). However, the computed RR performing ability (the shape based on the results of RR test) implied it depended on the amount of exposed proton. Based on the comparison between the results of the control (Fig. 3 A) and 30cGy-exposed groups (Fig. 3 B), both showed little difference in the coordinated motor control by the proton exposure in both short- ( 7 days). On the other hand, the exposure of 100 cGy (Fig. 3 C) reduced the performing ability until approximately 3 weeks after the exposure, increasing the computed area for the ability. The functional decline in the motional coordination was observed only in 100cGy-exposed group, and its recovery was initiated in 3 weeks. Interestingly, both the control and 30cGy-exposed groups similarly maintained the RR performing ability until up to 3 weeks, and it gradually declined, expanding the areas. The behavioral markers of the open field (OPF) test were mainly analyzed in the form of a changing rate (slopes on the data) in time (week), and they indicated the functional alteration was induced by the proton exposure (Fig. 4 ). The control group showed its moving distance and resting time increased while there was little modification in the mean speed with or without rest (Fig. 4 A). The increase of the resting time was consistently observed in 30cGy- and 100cGy-exposed groups, but the mean speed of both groups decreased no matter what the resting time existed (Fig. 4 B & 4 C). The results by the regression rates signified some detailed relations between the proton exposure and the behavioral patterns in OPF test. First, the resting times of both proton-exposed groups had a larger increasing pattern (9.11 and 10.81 sec/week for 30cGy- and 100cGy-exposed group, respectively), compared with that of the control (3.24 sec/week). Even though all groups showed an increased resting time, the tendency was strong in the proton-exposed groups, which suggested the proton exposure affected the animals’ moving pattern. Second, the total moving distance was also influenced by the proton-exposure. According to their regression rates, the moving distance of both exposed groups decreased, and the tendency in 100cGy-exposed group (-1.82 meter/week) was stronger than that in 30cGy-exposed group (-1.47 meter/week). In addition, that of the control group showed an opposite pattern (0.55 meter/week) from those of the proton-exposed groups in the moving distance. Thus, the behavioral pattern of the moving distance was expected to be modified by the amount of proton. Third, a similar consequence was noticed in the analysis of the mean speed with or without rest. The tendency of mean speed in the control group was contrary to those in the proton-exposed groups. However, the altering tendency in 100cGy-exposed group was the strongest while that in the control group was the weakest as shown in the consequence of the moving distance. As shown, the regression analysis demonstrated the behavior patterns was disturbed by the proton exposure, and the exposed amount of proton was critical to aggravate the given behavioral markers. The levels of fear/anxiety-like behavior (FAB) were assessed using the durations of in- and out-of-center, which was defined by the specified imaginary square (50 x 50 cm 2 ) in the middle of the open field (1 x 1 m 2 ) (see Fear/Anxiety-like Behavior Test ). In the control group, the duration out of center showed little difference (p > 0.01, t-test) as well as that in the center (p > 0.02, t-test) (Fig. 5 A). The 30cGy- and the 100cGy-exposed animals showed that the proton effect on the level of FAB was inconsistent, producing a significant or insignificant consequences (p 0.012, t-test) (Fig. 5 B & 5 C). Thus, the FAB levels based on the given periods rarely showed a conclusive result although the changing levels oscillated widely as time advanced. The most relevant example was the FAB levels of 100cGy-exposed group in the center (Fig. 5 C). The initial levels up to 4 weeks showed little difference (p > 0.023, t-test), but the oscillation grew bigger as time passed (p < 0.0064, t-test). On the other hand, the overall averages (dotted line) of FAB level, which covered the whole period, indicated that the effect of proton exposure depended on its amount. The overall average of out-of-center decreased as the exposed amount of proton increased, and that of in-center increased, suggesting the animal’s fear/anxiety-like behavior was weakened after the proton exposure. Few behavioral markers based on NOR test showed the proton-induced cognitive malfunction (Fig. 6 ). The principal consequence in NOR test was determined by the approaching time to the new object and the staying with it. However, the repeated test throughout 12 weeks indicated the numbers of entry to the old (light gray) and new zones (black) were statistically insignificant in the proton-exposed groups (p > 0.1, ANOVA) as well as the control group (p = 0.2, ANOVA) (Fig. 6 A). Similarly, the analysis of staying time implied little difference in each zone, providing no significance in all groups (p > 0.09, ANOVA) (Fig. 6 B). The comparisons of the number of entries to new zone among the different groups also showed no significance (p > 0.21, ANOVA) as well as those of the staying time to new zones (p > 0.87, ANOVA). Discussion The study of cognition has attracted an attention as one of the key topics in space research. Due to the increased staying time in space, the dysfunctional responses are commonly observed, and one of the most critical functions is cognition because of its direct relation to space mission performances [ 31 – 33 ]. Especially, the accumulated results demonstrated that the proton exposure produced some functional alteration in cognition by damaging the cells and the neurons in the central nervous system [ 21 , 22 , 24 – 26 ]. While the radiation-induced cognitive effects have been identified in various molecules, cells, and neurons, their relevant behavioral responses were inconsistent to show the cognitive alterations. According to the experiment with heavy ions (Fe, H, O, Ti, and Si), the discrimination index of novel object recognition slightly shaped a “W” form under the exposed amounts ranged between 0 (control) and 2Gy ( 12 C, 290 MeV/u) [ 34 , 35 ], suggesting ambiguous consequences after radiation. Another study using a proton exposure (up to 4Gy, 75-95cGy/min) also showed the inconsistency of cognitive behaviors based on open field test and rotarod [ 36 ]. Under the different exposed amounts of proton (3 & 4Gy), the analyzed behavioral parameters, such as the beam breaks, the staying time in field center or edge, and the number of rearing, hardly showed the cognition-related behavioral effect by the proton exposure. On the other hand, an investigation using proton (1Gy, 70 or 170MeV) and gamma exposure (1Gy, 60 Co) demonstrated the radiational effects on the FAB level, the rearing and freezing responses as well as some morphological changes in Hippocampus and abnormal formation of amyloid plaques [ 37 ]. As shown in the previous studies, the radiation-induced behavioral dysfunction is still elusive to demonstrate its direct relation to cognitive decline. Current study revisited some of widely adopted cognition-related behavioral tests to examine the proton-induced cognitive decline at the behavioral level. Moreover, even a low amount (30cGy) of proton might cause a recognizable behavioral consequence, which was known as a possible radiational amount for an astronaut (approx. 30cGy/year in International Space Station; approx. 40cGy/year on the lunar surface; approx. 60cGy/round-trip to Mars) [ 38 – 40 ]. To answer the raised question, we performed some analytical computations based on cognitive behaviors in time. In general, open field test (OPF) assesses the level of fear or anxiety-like behavior as well as locomotion [ 41 ]. In the computation, the durational effect was applied on the initial intention of OPF to show the proceeding changes in the moving distance and the resting time, and their results indicated the exposure to proton affected the selected behavioral parameters with time procedure (Fig. 4 ). The noted observation was maintained no matter how much amount of proton was applied, suggesting that the low amount of proton (30cGy) could cause the behavioral alteration based on the computed behavioral markers. The test of FAB level agreed with the proton-induced cognitive decline by low amount of proton. Even though the consistency in the responding activities was weak, the overall behavioral marker clearly represented the unstable condition of fear-related emotion as the time went (Fig. 5 ). Both behavioral markers demonstrated the low amount of proton affected some cognitive behaviors, such as FAB level and the moving responses. Unlike previous studies [ 42 – 44 ], no clear cognitive decline was identified in the test of novel object recognition (Fig. 6 ). The difference in the number of entries to old and new areas as well as background was unnoticeable depending on the amount of proton, and the duration since the exposure caused no significance in the responses. Other parameters, such as staying time in the specific areas, the resting time, and the mean speed at the areas, also indicated that there were no distinguished based on the amount of proton or the lapse of time since the exposure. Conclusion This study examined the proton-induced impacts on the animals’ cognitive behaviors, which were used to generate some practical behavioral markers. Our quantitative results were assessed using three types of cognitive behavior and one type of motor coordination test, and their computational analysis contributed to demonstrate the relation between the proton exposure and the cognitive alteration. The cognitive abnormality by the single proton exposure was identified by the computed behavioral markers, and the markers unveiled the effects by a low amount of proton (30 cGy), which was unrevealed by the raw data. Also, the behavioral markers demonstrated the relation between the cognitive decline and the amount of proton, and the functional decline increased as the amount of proton increased. In conclusion, the current analysis suggested that the proton exposure impairs the cognitive behaviors by reducing the motional distance and the mean speed and increasing the resting time. The patterns of these behavioral performance were quantitatively assessed by the computational analysis, and it eventually showed the effects of proton on the cognitive decline. Declarations Author contributions GT performed the study plan, data collection, and data analysis. GT also wrote the initial manuscript, and he revised it for the final manuscript. KS provided an insightful idea and fundings for this study. Funding This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded partially by the Ministry of Education and Korea Health Industry Development Institute (KHIDI) (2018R1A6A1A03025523 & RS-2023-00266209). Availability of data and materials The datasets generated during the current study are available from the corresponding author on reasonable request. Conflict of interest The authors declare no conficts of interest or competing interests. Ethical approval Animal Ethics Committee at Inha University (InCheon, Korea) approved all procedures and principles of laboratory animal care in this study (INHA 220822-835). References Patel ZS, Brunstetter TJ, Tarver WJ, Whitmire AM, Zwart SR, Smith SM, Huff JL (2020) Red risks for a journey to the red planet: The highest priority human health risks for a mission to Mars. npj Microgravity. 6:33. https://doi.org/10.1038/s41526-020-00124-6 . 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Process. 22(Suppl 1):S105-S114. https://doi.org/10.1007/s10339-021-01050-5 . McGregor HR, Hupfeld KE, Pasternak O, Beltran NE, De Dios YE, Bloomberg JJ, Wood SJ, Mulavara AP, Riascos RF, Reuter-Lorenz PA, Seidler RD (2023) Impacts of spaceflight experience on human brain structure. Sci. Rep. 13:7878. https://doi.org/10.1038/s41598-023-33331-8 . Desai RI, Limoli CL, Stark CEL, Stark SM (2022) Impact of spaceflight stressors on behavior and cognition: A molecular, neurochemical, and neurobiological perspective. Neurosci. Biobehav. Rev. 138:104676. https://doi.org/10.1016/j.neubiorev.2022.104676 . Rabin BM, Shukitt-Hale B, Joseph JA, Carrihill-Knoll KL, Carey AN, Cheng V (2007) Relative effectiveness of different particles and energies in disrupting behavioral performance. Radiat. Environ. Biophys. 46:173–177. https://doi.org/10.1007/s00411-006-0071-2 . Rabin BM, Carrihill-Knoll KL, Shukitt-Hale B (2011) Operant responding following exposure to HZE particles and its relationship to particle energy and linear energy transfer. Adv. Space Res. 48:370–377. https://doi.org/10.1016/j.asr.2011.03.008 . Pecaut MJ, Haerich P, Zuccarelli CN, Smith AL, Zendejas ED, Nelson GA (2002) Behavioral consequences of radiation exposure to simulated space radiation in the C57BL/6 mouse: Open field, rotarod, and acoustic startle. Cog. Affect. & Behav. Neurosci. 2(4):329–340. https://doi.org/10.3758/CABN.2.4.329 . Severyukhin YS, Lalkovicova M, Utina DM, Lyakhova KN, Kolesnikova IA, Ermolaeva ME, Molokanov AG, Gaevsky VN, Komarov DA, Krasavin EA (2023) Comparative Analysis of Behavioral Reactions and Morphological Changes in the Rat Brain After Exposure to Ionizing Radiation with Different Physical Characteristics. Cell. Mole. Neurobiol. 43:339–353. https://doi.org/10.1007/s10571-021-01187-z . Zeitlin C, Hassler DM, Cucinotta FA, et al. (2013) Measurements of energetic particle radiation in transit to Mars on the Mars Science Laboratory. Science. 340:1080–1084. https://doi.org/10.1126/science. 1235989 . Naito M, Hasebe N, Shikishima M, et al. (2020) Radiation dose and its protection in the Moon from galactic cosmic rays and solar energetic particles: at the lunar surface and in a lava tube. J. Radiol. Prot. 40:947–961. https://doi.org/10.1088/1361-6498/abb120 . Kodaira S, Naito M, Uchihori Y, Hashimoto H, Yano H, Yamagish A (2021) Space Radiation Dosimetry at the Exposure Facility of the International Space Station for the Tanpopo Mission. Astrobiol. 21:12. https://doi.org/10.1089/ast.2020.2427 . Seibenhener ML, Wooten MC (2015) Use of the open field maze to measure locomotor and anxiety-like behavior in mice. J. Vis. Exp. 96:52434. https://doi.org/10.3791/52434 . Krotkova OA, Kuleva AY, Galkin MV, Kaverina MY, Strunina YV, Danilov GV (2021) Memory Modulation Factors in Hippocampus Exposed to Radiation. Sovrem Tekhnologii Med. 13(4): 6–13. https://doi.org/10.17691/stm2021.13.4.01 . Wang K, Lu JM, Xing ZH, Zhao QH, Hu LQ, Xue L, Zhang J, Mei YA (2017) Effect of 1.8 GHz radiofrequency electromagnetic radiation on novel object associative recognition memory in mice. Sci. Rep. 7: 44521. https://doi.org/10.1038/srep44521 . Cacao E, Cucinotta FA (2019) Meta-analysis of cognitive performance by novel object recognition after proton and heavy ion exposures. Rad. Res. 192(5):463–472. https://doi.org/10.1667/RR15419.1 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3893200","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":269620398,"identity":"53e86d2f-730b-4b7b-92f1-0d89854873cc","order_by":0,"name":"Gyutae Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuklEQVRIiWNgGAWjYBACCWYg8cGAIQHCPUCkFsYZpGkBYmYeBlK0SLZzJ7+2KbiTx9/A/PADw5l7hLVIM/Nus84xeFYscYDNWILhRjFhLXJALcY5BocTGw4wmAEDIoFILRZALfMPsH8jTgvQYZsfMwC1bDjAA7TlBhFaJJt5tzH2ALVsPMxTLJFwhggtEufPbv7w48/hxHnH2zd++HCMCC1AwCYBpkDJgDgNQLUfiFQ4CkbBKBgFIxUAAN5DOXBDMuU7AAAAAElFTkSuQmCC","orcid":"","institution":"Inha University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Gyutae","middleName":"","lastName":"Kim","suffix":""},{"id":269620399,"identity":"a06dcd9f-831d-4dca-aa8d-29e9e3562d4f","order_by":1,"name":"Kyu-Sung Kim","email":"","orcid":"","institution":"Inha University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kyu-Sung","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2024-01-24 06:48:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3893200/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3893200/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50366199,"identity":"f5e4965d-dfa6-4351-bb64-6b298086421b","added_by":"auto","created_at":"2024-01-30 11:45:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67632,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental schedule. Four main tests (rota-rod, open field, fear test and novel object) were performed in the given days. Animals performed the rota-rod and 3 cognitive behavioral tests (open field test, fear test \u0026amp; novel object recognition) before and after the proton exposure. The relevant effects were generally analyzed by comparing with the results of the control group. For each test, a different number of animals were selected out of the total population (N=28).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3893200/v1/670c0a300c9c34894e984ff0.png"},{"id":50366075,"identity":"e1bc27d2-5776-4ece-b63b-df3aaa218ca0","added_by":"auto","created_at":"2024-01-30 11:37:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":16779,"visible":true,"origin":"","legend":"\u003cp\u003eChange of weight. (A) All measured weights (in black, in blue, and in red for control, 30cGy-exposed, and 100cGy-exposed groups, respectively) were presented with the referenced weight (in gray). (B) Specific weights in the early (up to 7 days), 4 weeks, and 12 weeks were displayed with their regressions, which were generated in the 2\u003csup\u003end\u003c/sup\u003e order polynomial function. Total 21 out of 28 animals (75% of total population) were involved for the analysis of weight.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3893200/v1/eb4b14ca6b59f166425260f8.png"},{"id":50366074,"identity":"669967f9-2a9d-4b33-984d-4e1cfe1bdff3","added_by":"auto","created_at":"2024-01-30 11:37:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":27959,"visible":true,"origin":"","legend":"\u003cp\u003eMeasured and simulated performance pattern of rota-rod test. As designed, the animals were expected to stay on the rolling rod for 180-200 seconds to identify the normality of motional coordination. All plots were divided into 2 separated periods, such as before and after proton exposure, and the day of “0” meant the day of proton exposure. (A) Rota-rod (RR) performance pattern of control group. (B) RR performance pattern of 30cGy-exposed group (C) RR performance pattern of 100cGy-exposed group. Total 18 out of 28 animals (64.3% of total population) were involved for the analysis of performance, and 10, 4, and 4 animals were used for control, 30cGy-exposed, and 100cGy-exposed group, respectively.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3893200/v1/e5a35944517b0a61fe375d75.png"},{"id":50366078,"identity":"40e554e0-3326-4296-98cc-057fdd3046f6","added_by":"auto","created_at":"2024-01-30 11:37:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30858,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral markers of open field test. Three indicators were analyzed, such as total distance of motion, mean speed, and resting time. (A) The indicators of control group. The indicator of total distance was generated by a linear regression on the averaged distance at the given time (week). At each time, the mean speeds with and without rest were presented. At the bottom, the resting times at the periods was presented with their regression. (B) The indicators of 30cGy-exposed group (C) The indicators of 100cGy-exposed group. In (B) and (C), the format of plots was the same as (A). Total 14 out of 28 animals (50% of total population) were used for the behavioral markers of open field test, and 4, 5, and 5 animals were used for control, 30cGy-exposed, and 100cGy-exposed group, respectively.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3893200/v1/dab5e306fd0628a3dcee8045.png"},{"id":50366674,"identity":"4b34c733-8c3a-42a6-8671-ff564a7f69fd","added_by":"auto","created_at":"2024-01-30 11:53:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":37603,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral markers of fear test. In- \u0026amp; Out-of-Centers were defined, and the remaining duration (sec) in each area was measured. Three groups (\u003cstrong\u003eA\u003c/strong\u003e: control, \u003cstrong\u003eB\u003c/strong\u003e: 30cGy-exposed \u0026amp; \u003cstrong\u003eC\u003c/strong\u003e: 100cGy-exposed groups) with 5 animals were independently presented depending on the periods (1,3,4,5,8,11, and 12 weeks) after proton exposure. Individual durations (open circles) and their averaged durations (closed circles) were presented at the given periods. The overall average (dotted line) in each group was used to assess the exposing effects by different amounts of proton.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3893200/v1/629829bb2a432d632961a122.png"},{"id":50366079,"identity":"bbb8041f-a82d-4dde-9fe7-ab3794316549","added_by":"auto","created_at":"2024-01-30 11:37:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":277852,"visible":true,"origin":"","legend":"\u003cp\u003eBehavioral markers of novel object recognition. Two indicators were mainly analyzed, such as number of entry and staying time in three defined zones. The separated zones were an old zone with a familiar object, a new zone with a novel object, and a background with no object. Both indicators were presented, depending on the given zones, such as background (in dark gray), old (in light gray), and new zone (in black). Due to the different number and time, the indicators were displayed in percentages (%) for a consistent representation. (A) Number of entries to the zones by 3 groups in 1\u003csup\u003est\u003c/sup\u003e, 3\u003csup\u003erd\u003c/sup\u003e, 4\u003csup\u003eth\u003c/sup\u003e, 5\u003csup\u003eth\u003c/sup\u003e, 8\u003csup\u003eth\u003c/sup\u003e, 11\u003csup\u003eth\u003c/sup\u003e, and 12\u003csup\u003eth\u003c/sup\u003e weeks. (B) Staying time in the zones by 3 groups in 1\u003csup\u003est\u003c/sup\u003e, 3\u003csup\u003erd\u003c/sup\u003e, 4\u003csup\u003eth\u003c/sup\u003e, 5\u003csup\u003eth\u003c/sup\u003e, 8\u003csup\u003eth\u003c/sup\u003e, 11\u003csup\u003eth\u003c/sup\u003e, and 12\u003csup\u003eth\u003c/sup\u003e weeks. Total 18 out of 28 animals (64.3% of total population) were used for the behavioral markers of novel object recognition, and 10, 4, and 4 animals were used for control, 30cGy-exposed, and 100cGy-exposed group, respectively.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3893200/v1/16686224c624e75e9990efb9.png"},{"id":50585285,"identity":"9d79b007-5e1d-4a41-a9c4-cfeee5d325ee","added_by":"auto","created_at":"2024-02-02 21:52:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1032012,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3893200/v1/840988d6-163a-464b-a1ac-15897cc5af9d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Proton-induced Behavioral Alterations approved Cognitive Dysfunction","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCurrent NASA\u0026rsquo;s projects have issued 5 hazardous factors, such as space radiation, gravity alteration, closed environments, isolation, and distance from Earth during space exploration. These potential risks induce various structural and functional abnormalities in molecules, cells, and neurons as well as organ systems [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Of the addressed factors, space radiation is one of the most influential risks to damage a wide range of biological levels from DNA [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] to central nervous system [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Space radiation is composed of various types of energies and linear energy transfer (LTE), and most destructive effects were known to be generated by the heavy ionic particles (1\u0026ndash;2%) while proton is a dominant type (up to 91%) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Due to the damage degrees, many previous studies have focused on the physiological and functional alterations by the heavy ionic particles, underestimating those by proton.\u003c/p\u003e \u003cp\u003eAt the early stage of space exploration, the amount of radiation exposure was limited, but its impacts have increased as space mission expanded to Mars (expected dose: 350mSv/year for 3 years) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In general, the heavy ions, such as hydrogen (\u003csup\u003e1\u003c/sup\u003eH\u003csup\u003e1\u003c/sup\u003e), carbon (\u003csup\u003e12\u003c/sup\u003eC\u003csup\u003e6\u003c/sup\u003e), silicone (\u003csup\u003e28\u003c/sup\u003eS\u003csup\u003e14\u003c/sup\u003e), titanium (\u003csup\u003e48\u003c/sup\u003eTi\u003csup\u003e22\u003c/sup\u003e), and iron (\u003csup\u003e56\u003c/sup\u003eFe\u003csup\u003e26\u003c/sup\u003e), damage DNA by producing lesion-specific enzymes to break its double-strand [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Once the series of damages are initiated, the radiation-induced effects sequentially expand to the damages at upper biological levels, like chromosomal aberrations [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], cell kinetics [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], mitotic death [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], peripheral blood lymphocytes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, the triggered damages affect the functions of central nervous system as well as inner organs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. As shown in these previous studies, various biological targets have been examined to understand the radiation-induced effects, and the relevant interest is moving to the function of cognition as the staying duration increased in Space [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProton, which is the dominant type in space radiation as mentioned, has been mainly used for the medical purpose, specifically cancer therapy [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The repeated treatment by proton exposure raised the concerns of cognitive impairment, and some studies demonstrated the relation between the proton exposure and the cognition-related functional decline [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. However, the cognition-related behavioral alteration as the functional endpoint after proton exposure is still ambiguous while the neurochemical [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], neurophysiological [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and functional images [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] have indicated the proton-induced damages in the brain. Thus, many studies emphasized various proton-induced molecular and neuronal damages with no direct equivocal behavioral characteristics. Especially, the biological consequence under a low amount of proton (\u0026lt;\u0026thinsp;1Gy) has been rarely investigated while some heavy ions induced a loss of bone mass by the same amount [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, we proposed some behavioral markers, which were computed based on the moving distance, the mean speed, and the resting time in open field test, to assess the behavioral alteration after the proton exposure. In addition, the behavioral tests, such as novel object recognition and rota-rod tests, were also adopted to estimate the functional changes of cognition, and their own markers were generated to examine their superiority to those in open field test. Based on computed behavioral markers, we identified the cognition-related changes in open field test, and the suggested analysis quantitatively distinguished the behavioral responses to a low amount of proton (30cGy) from normal activities. Considering the characteristics of the radiation-induced effects, which gradually spread over the biological systems during a long time, the relevant impacts needed to be analyzed in respect to time, and our current analysis adopted the hypothesis in the time basis. This helped to generalize the proton-induced behavioral effects, and it also demonstrated the time relation between the cognitive behavioral responses and the amount of radiation exposure.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e All procedures and principles of laboratory animal care were approved by the Animal Ethics Committee at Inha University (INHA 220822-835). Total 28 rats (Sprague Dawley, M) were used in this study, and the animals were divided into 3 groups (control, 30cGy-, and 1Gy-exposed animals) based on the amount of proton exposure. Out of total population, some selective animals were used for the assessment of the changes in weight, rota-rod test, open field test, and novel object recognition at scheduled periods for behavioral markers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All animals were housed in the controlled environments, such as stable temperature (22\u0026ndash;25℃), humidity (40\u0026ndash;60%), ventilation (10\u0026ndash;15 times/hour), static pressure difference (\u0026gt;\u0026thinsp;5mmAq), noise level (\u0026lt;\u0026thinsp;60dB) and a 12:12 hours light-dark cycle except during the experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal preparation \u0026amp; Proton Exposure\u003c/h2\u003e \u003cp\u003eBefore the relocation for the proton exposure, some selected animals (n\u0026thinsp;=\u0026thinsp;21) were initially trained to attain a prerequisite level (180\u0026ndash;200 sec) of the motional balance on the rota-rod test. Once the animals\u0026rsquo; balance level was ready, the animals were relocated for the proton beam exposure at Korea Multi-purpose Accelerator Complex (KOMAC, KyungJu, South Korea). The animals were moved by a ground transportation (approximately 350 km) one day before the proton exposure for the animals\u0026rsquo; stability. After 24-hours stabilization, an animal was anesthetized by an intramuscular injection of a mixed solution of Ketamine (1 \u0026micro;l/g) and Xylazine (0.33 \u0026micro;l/g), and it was placed in the self-produced holding frame. The frame with an animal was positioned at about 1.5 m away from the beam outlet. Using a 100-MeV beam energy, single proton exposure was conducted. The effective area of exposure was 10\u0026times;10 cm\u003csup\u003e2\u003c/sup\u003e, and its uniformity was 95.93%. The beam exposure was completed by multiple beam pulses (energy per pulse: 0.0026\u0026ndash;0.0035 Gy/pulse), and two final doses were 30 cGy and 1 Gy (error rate\u0026thinsp;\u0026lt;\u0026thinsp;2.5%). Initially, the weight change and skin damage were estimated the stresses of the beam exposure and the long transportation, and these changes were again inspected at the sacrificing period (72 hours, 4 weeks, and 3 months) for a molecular examination.\u003c/p\u003e \u003cp\u003eThe weight change of animals was examined based on the comparison with the normal weight curve (Hilltop Lab Animals, Inc., Pennsylvania, US). On the normal weight chart, all animals\u0026rsquo; weights were overlapped to identify the effects of the long-distance transportation and radiation exposure. The normality of animals\u0026rsquo; condition was confirmed by the weights. Especially, any weights below the normal one at a given time were counted as the result by the long transportation or the proton exposure. Considering that the radiation-related symptoms generally appeared in 1\u0026ndash;2 weeks later since exposure, the early change in the weights was assumed as the consequence by the long-term transportation for the experiment of proton exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRota-Rod test\u003c/h2\u003e \u003cp\u003eRota-rod (RR) test estimated coordinated motional balance after the proton exposure. Some selected animals (n\u0026thinsp;=\u0026thinsp;18) walked on the rolling rod with a constant speed (34 meter/min), and one RR test lasted for approximately 3 minutes, which repeated in 5 times with a five-minute break. The test was ceased if the animal changed its walking direction oppositely or the animal was just hanging the rolling rod with no walk. The test was generally conducted at 3, 7, and 10 days before the exposure, and at 3, 7, 28, 42, 56, 70, 84, 86, and 88 days after the proton exposure. Before the relocation for the proton exposure, all animals were trained to reach a required walking level, which was ranged between 180 and 200 seconds, and the applied regulations for the test were the same before and after the exposure. The divided groups based on the amount of proton exposure performed the RR test on the scheduled days, and the attained time on the rod was used to assess the motional balance. Once the animal reached the required time for the test, the RR test was ceased.\u003c/p\u003e \u003cp\u003eThe overall results of RR test from the different animal groups were separately plotted in a closed form containing all the measurements to predict their changing patterns of test performance. Using a custom-written code (MATLAB, MathWorks, US), all the measurements in time (days) were presented up to 3 months, and a smoothed area based on the measurements was calculated. The shape of the computed area showed the predicted pattern in each animal group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eOpen Field test\u003c/h2\u003e \u003cp\u003eUsing 14 out of total animals, open field (OPF) test was designed for the quantitative evaluation of animal\u0026rsquo;s locomotor activity as well as the animals\u0026rsquo; moving pattern. OPF test was performed at 7, 14, 21, 28, 35, 56, 77, and 84 days after the proton exposure. Specific parameters in OPF test were measured, such as total distance, resting time, and mean speed with or without resting, using a video tracking system (SMART 3.0, Harvard/PANLab, US). During a 10-minute (600 seconds) test, animals\u0026rsquo; movements were detected and calculated in the defined area, so-called the zone of a background (1 x 1 m\u003csup\u003e2\u003c/sup\u003e), and the total distance was measured based on the automated detection. Distance in zone showed the total moving length in the given area. The relevant results were presented by their means and standard deviation (STD) (m) at the planned periods. A linear regression was applied on the means of distances in time to assess the proton effects. The resting (immobile) time was defined by a certain speed (\u0026lt;\u0026thinsp;0.5 cm/sec) which indicated the animal showed little movement during the detection. Once the total distance and resting time were confirmed, the mean speed with or without resting time was calculated as follows;\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$${Mean Speed}_{without resting}=\\frac{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l} \\text{D}\\text{i}\\text{s}\\text{t}\\text{a}\\text{n}\\text{c}\\text{e}}{600-\\text{r}\\text{e}\\text{s}\\text{t}\\text{i}\\text{n}\\text{g} \\text{t}\\text{i}\\text{m}\\text{e}}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$${Mean Speed}_{with resting}=\\frac{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l} \\text{D}\\text{i}\\text{s}\\text{t}\\text{a}\\text{n}\\text{c}\\text{e}}{600}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eCombining with total moving distance, the mean speed (cm/sec) was presented in bar charts with or without the resting time.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFear/Anxiety-like Behavior Test\u003c/h2\u003e \u003cp\u003eFifteen animals out of the total population were used to assess the levels of fear/anxiety-like behavior. An animal freely moved in the same area of open field (1 x 1 m\u003csup\u003e2\u003c/sup\u003e), and all movements were recorded. Using the recorded movie (10 frames / sec), the duration (sec) of in- or out-of-center was measured, and the time duration in each specified area was mainly used to estimate the level of fear. Each animal\u0026rsquo;s location in either in-center or out-of-center was determined by an imaginary square (50 x 50 cm\u003csup\u003e2\u003c/sup\u003e), positioned at the center of the open field. Thus, the condition of in-center implied that the animal located in the imaginary square, and that of out-of-center did that the location of animal was out of the square. At the selected periods (1,3,4,5,8,11, and 12 weeks after proton exposure), the durations of in- and out-of-center were measured, and the individual (open circle) and the averaged time (\u0026plusmn;\u0026thinsp;standard deviation) (closed circle) were presented. Using the averages at the periods, the overall means (dotted lines) of in- and out-of-center were computed to show the level of fear/anxiety-like behavior by the different amount of proton exposure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eNovel Object Recognition\u003c/h2\u003e \u003cp\u003eNovel object recognition (NOR) test assessed an animal\u0026rsquo;s ability to identify a newly presented object, which the cognitive function was involved with. The test initially began by placing an animal in a squared area (1 x 1 m\u003csup\u003e2\u003c/sup\u003e) with two same objects (aluminium-material, 245ml in volume, 9 x 6.5 cm\u003csup\u003e2\u003c/sup\u003e in height x diameter). For a given time (10 minutes), the animal was exposed to these objects, and it became eventually accustomed to them, so called familiar objects (FO) (the first searching session). These objects were located at randomized two corners in the squared area, and the animal was allowed to freely search the zones, physically contacting two FOs. The identification of an object was defined as the time which the animal was located at around the objects (generally, the defined zone within 4 cm away from the surface of the object). Once the adapting time was completed for the familiarity to FOs, the animal took a rest (60 minutes). After the break, one of FOs was replaced by a new object (glass-material, 155ml in volume, 14 x 3.5 cm\u003csup\u003e2\u003c/sup\u003e in height x diameter), and the animal again underwent the second searching session (10 minutes). Some selected animals (n\u0026thinsp;=\u0026thinsp;18) were used for this test. As explained, the animal\u0026rsquo;s cognitive function was measured by the staying time in the defined zone. As the new object was recognized by the animal, the animal was expected to stay in the zone with the new object longer than that with FO. For a separate analysis, 3 zones were defined; the zone with FO as old zone, that with new object as new zone, and the background with no object. The main behavioral markers in NOR test were the number of entries to zone and the staying time in zone, which estimated the recognition of a newly presented object.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eFor the statistical evaluation of results, 2-sampled t-test and 2-way analysis of variance (ANOVA) were adopted for the analysis of weights and NOR test, respectively. The weights were tested if the results of two proton-exposed groups were significantly different from that of control group, and the same test was applied to identify any significance at multiple periods of time (less than 1st, 4th, and 12th week). The results of NOR was examined based on the number of entry and staying time with a given time intervals (1st, 3rd, 4th, 5th, 8th, 11th, and 12th week). At these periods of time after the proton exposure, the number of entry and staying time in the defined zones were calculated based on the relative percentages. Using these computed values, the statistical analysis by ANOVA was performed. The level of significance was 0.01, and all statistical analysis were conducted by a user-written code in MATLAB (MathWorks, US).\u003c/p\u003e \u003cp\u003eStatistical comparison in fear/anxiety-like behavior test was conducted by t-test (significance: 0.01). For each group (control, 30cGy- \u0026amp; 100cGy-exposed groups), 5 animals\u0026rsquo; locations in or out of center were measured at the given period (1st, 3rd, 4th, 5th, 8th, 11th, and 12th week), and these individual durations at the periods were compared to show how much the exposed proton affected the function of fear/anxiety-like behavior by lapse of time.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAnimals were trained to be familiar with the rota-rod (RR) test, and they repeated the test until they reached the required level of performance before the transportation for the proton exposure (see \u003cb\u003eRota- Rod test\u003c/b\u003e). Once the animals were ready for the RR test, they were relocated to the laboratory in Korea Atomic Energy Research Institute (KOMAC, KyungJu, South Korea) one-day before the proton experiment. After the exposure was completed, the animals were transported back to Inha lab. The change of weights indicated the effects of the long transportation and the proton exposure were minor (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Comparing with the referenced weights in days (band in gray), no group showed any significant difference in the weights (p\u0026thinsp;\u0026gt;\u0026thinsp;0.059, 2-sampled t-test), except that between the control and 100 cGy-exposed group at 12th week (p\u0026thinsp;=\u0026thinsp;0.042, 2-sampled t-test). Thus, the result indicated no early effect by the proton exposure as well as by the long transportation, but the amount of proton, 100 cGy, generated a proton-induced effect in a long-term period (\u0026gt;\u0026thinsp;12 weeks). The overall changes in animals\u0026rsquo; weights up to 12 weeks also supported this inference, and the weights of all groups remained in the expected range of the referenced weights (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). On the other hand, the regressions using the weights at three periods (3, 28, and 84 days after the exposure) anticipated an early effect by the long transportation and the proton exposure, but the increased difference between the control and proton-exposed groups indicated the effect was mainly caused by the proton exposure instead by the long transportation which was limited at the early period (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Therefore, the difference of weights between the control and the proton-exposed groups was resulted by the proton exposure while that by the long transportation was limited.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAll animals attained the expected level of the performance in RR test within a week, and the relevant capability was generally maintained even after the proton exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, the computed RR performing ability (the shape based on the results of RR test) implied it depended on the amount of exposed proton. Based on the comparison between the results of the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and 30cGy-exposed groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), both showed little difference in the coordinated motor control by the proton exposure in both short- (\u0026lt;\u0026thinsp;7 days) and long-term (\u0026gt;\u0026thinsp;7 days). On the other hand, the exposure of 100 cGy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) reduced the performing ability until approximately 3 weeks after the exposure, increasing the computed area for the ability. The functional decline in the motional coordination was observed only in 100cGy-exposed group, and its recovery was initiated in 3 weeks. Interestingly, both the control and 30cGy-exposed groups similarly maintained the RR performing ability until up to 3 weeks, and it gradually declined, expanding the areas.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe behavioral markers of the open field (OPF) test were mainly analyzed in the form of a changing rate (slopes on the data) in time (week), and they indicated the functional alteration was induced by the proton exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The control group showed its moving distance and resting time increased while there was little modification in the mean speed with or without rest (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The increase of the resting time was consistently observed in 30cGy- and 100cGy-exposed groups, but the mean speed of both groups decreased no matter what the resting time existed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u0026amp; \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The results by the regression rates signified some detailed relations between the proton exposure and the behavioral patterns in OPF test. First, the resting times of both proton-exposed groups had a larger increasing pattern (9.11 and 10.81 sec/week for 30cGy- and 100cGy-exposed group, respectively), compared with that of the control (3.24 sec/week). Even though all groups showed an increased resting time, the tendency was strong in the proton-exposed groups, which suggested the proton exposure affected the animals\u0026rsquo; moving pattern. Second, the total moving distance was also influenced by the proton-exposure. According to their regression rates, the moving distance of both exposed groups decreased, and the tendency in 100cGy-exposed group (-1.82 meter/week) was stronger than that in 30cGy-exposed group (-1.47 meter/week). In addition, that of the control group showed an opposite pattern (0.55 meter/week) from those of the proton-exposed groups in the moving distance. Thus, the behavioral pattern of the moving distance was expected to be modified by the amount of proton. Third, a similar consequence was noticed in the analysis of the mean speed with or without rest. The tendency of mean speed in the control group was contrary to those in the proton-exposed groups. However, the altering tendency in 100cGy-exposed group was the strongest while that in the control group was the weakest as shown in the consequence of the moving distance. As shown, the regression analysis demonstrated the behavior patterns was disturbed by the proton exposure, and the exposed amount of proton was critical to aggravate the given behavioral markers.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe levels of fear/anxiety-like behavior (FAB) were assessed using the durations of in- and out-of-center, which was defined by the specified imaginary square (50 x 50 cm\u003csup\u003e2\u003c/sup\u003e) in the middle of the open field (1 x 1 m\u003csup\u003e2\u003c/sup\u003e) (see \u003cb\u003eFear/Anxiety-like Behavior Test\u003c/b\u003e). In the control group, the duration out of center showed little difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.01, t-test) as well as that in the center (p\u0026thinsp;\u0026gt;\u0026thinsp;0.02, t-test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The 30cGy- and the 100cGy-exposed animals showed that the proton effect on the level of FAB was inconsistent, producing a significant or insignificant consequences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0064 or p\u0026thinsp;\u0026gt;\u0026thinsp;0.012, t-test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB \u0026amp; \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Thus, the FAB levels based on the given periods rarely showed a conclusive result although the changing levels oscillated widely as time advanced. The most relevant example was the FAB levels of 100cGy-exposed group in the center (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). The initial levels up to 4 weeks showed little difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.023, t-test), but the oscillation grew bigger as time passed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0064, t-test). On the other hand, the overall averages (dotted line) of FAB level, which covered the whole period, indicated that the effect of proton exposure depended on its amount. The overall average of out-of-center decreased as the exposed amount of proton increased, and that of in-center increased, suggesting the animal\u0026rsquo;s fear/anxiety-like behavior was weakened after the proton exposure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFew behavioral markers based on NOR test showed the proton-induced cognitive malfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The principal consequence in NOR test was determined by the approaching time to the new object and the staying with it. However, the repeated test throughout 12 weeks indicated the numbers of entry to the old (light gray) and new zones (black) were statistically insignificant in the proton-exposed groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.1, ANOVA) as well as the control group (p\u0026thinsp;=\u0026thinsp;0.2, ANOVA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Similarly, the analysis of staying time implied little difference in each zone, providing no significance in all groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.09, ANOVA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). The comparisons of the number of entries to new zone among the different groups also showed no significance (p\u0026thinsp;\u0026gt;\u0026thinsp;0.21, ANOVA) as well as those of the staying time to new zones (p\u0026thinsp;\u0026gt;\u0026thinsp;0.87, ANOVA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe study of cognition has attracted an attention as one of the key topics in space research. Due to the increased staying time in space, the dysfunctional responses are commonly observed, and one of the most critical functions is cognition because of its direct relation to space mission performances [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Especially, the accumulated results demonstrated that the proton exposure produced some functional alteration in cognition by damaging the cells and the neurons in the central nervous system [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. While the radiation-induced cognitive effects have been identified in various molecules, cells, and neurons, their relevant behavioral responses were inconsistent to show the cognitive alterations. According to the experiment with heavy ions (Fe, H, O, Ti, and Si), the discrimination index of novel object recognition slightly shaped a \u0026ldquo;W\u0026rdquo; form under the exposed amounts ranged between 0 (control) and 2Gy (\u003csup\u003e12\u003c/sup\u003eC, 290 MeV/u) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], suggesting ambiguous consequences after radiation. Another study using a proton exposure (up to 4Gy, 75-95cGy/min) also showed the inconsistency of cognitive behaviors based on open field test and rotarod [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Under the different exposed amounts of proton (3 \u0026amp; 4Gy), the analyzed behavioral parameters, such as the beam breaks, the staying time in field center or edge, and the number of rearing, hardly showed the cognition-related behavioral effect by the proton exposure. On the other hand, an investigation using proton (1Gy, 70 or 170MeV) and gamma exposure (1Gy, \u003csup\u003e60\u003c/sup\u003eCo) demonstrated the radiational effects on the FAB level, the rearing and freezing responses as well as some morphological changes in Hippocampus and abnormal formation of amyloid plaques [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. As shown in the previous studies, the radiation-induced behavioral dysfunction is still elusive to demonstrate its direct relation to cognitive decline.\u003c/p\u003e \u003cp\u003eCurrent study revisited some of widely adopted cognition-related behavioral tests to examine the proton-induced cognitive decline at the behavioral level. Moreover, even a low amount (30cGy) of proton might cause a recognizable behavioral consequence, which was known as a possible radiational amount for an astronaut (approx. 30cGy/year in International Space Station; approx. 40cGy/year on the lunar surface; approx. 60cGy/round-trip to Mars) [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. To answer the raised question, we performed some analytical computations based on cognitive behaviors in time.\u003c/p\u003e \u003cp\u003eIn general, open field test (OPF) assesses the level of fear or anxiety-like behavior as well as locomotion [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the computation, the durational effect was applied on the initial intention of OPF to show the proceeding changes in the moving distance and the resting time, and their results indicated the exposure to proton affected the selected behavioral parameters with time procedure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The noted observation was maintained no matter how much amount of proton was applied, suggesting that the low amount of proton (30cGy) could cause the behavioral alteration based on the computed behavioral markers. The test of FAB level agreed with the proton-induced cognitive decline by low amount of proton. Even though the consistency in the responding activities was weak, the overall behavioral marker clearly represented the unstable condition of fear-related emotion as the time went (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Both behavioral markers demonstrated the low amount of proton affected some cognitive behaviors, such as FAB level and the moving responses.\u003c/p\u003e \u003cp\u003eUnlike previous studies [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], no clear cognitive decline was identified in the test of novel object recognition (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The difference in the number of entries to old and new areas as well as background was unnoticeable depending on the amount of proton, and the duration since the exposure caused no significance in the responses. Other parameters, such as staying time in the specific areas, the resting time, and the mean speed at the areas, also indicated that there were no distinguished based on the amount of proton or the lapse of time since the exposure.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study examined the proton-induced impacts on the animals\u0026rsquo; cognitive behaviors, which were used to generate some practical behavioral markers. Our quantitative results were assessed using three types of cognitive behavior and one type of motor coordination test, and their computational analysis contributed to demonstrate the relation between the proton exposure and the cognitive alteration. The cognitive abnormality by the single proton exposure was identified by the computed behavioral markers, and the markers unveiled the effects by a low amount of proton (30 cGy), which was unrevealed by the raw data. Also, the behavioral markers demonstrated the relation between the cognitive decline and the amount of proton, and the functional decline increased as the amount of proton increased. In conclusion, the current analysis suggested that the proton exposure impairs the cognitive behaviors by reducing the motional distance and the mean speed and increasing the resting time. The patterns of these behavioral performance were quantitatively assessed by the computational analysis, and it eventually showed the effects of proton on the cognitive decline.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGT performed the study plan, data collection, and data analysis. GT also wrote the initial manuscript, and he revised it for the final manuscript. KS provided an insightful idea and fundings for this study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded partially by the Ministry of Education and Korea Health Industry Development Institute (KHIDI) (2018R1A6A1A03025523\u0026nbsp;\u0026amp; RS-2023-00266209).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during the current study are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conficts of interest or competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal Ethics Committee at Inha University (InCheon, Korea) approved all procedures and principles of laboratory animal care in this study (INHA 220822-835).\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePatel ZS, Brunstetter TJ, Tarver WJ, Whitmire AM, Zwart SR, Smith SM, Huff JL (2020) Red risks for a journey to the red planet: The highest priority human health risks for a mission to Mars. npj Microgravity. 6:33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41526-020-00124-6\u003c/span\u003e\u003cspan address=\"10.1038/s41526-020-00124-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHada M, Sutherland BM (2006) Spectrum of Complex DNA Damages Depends on the Incident Radiation. 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Res. 192(5):463\u0026ndash;472. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1667/RR15419.1\u003c/span\u003e\u003cspan address=\"10.1667/RR15419.1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cognition, Behavioral Response, Space Environment, Radiation, Hypergravity","lastPublishedDoi":"10.21203/rs.3.rs-3893200/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3893200/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA long-term space exploration faces extremely hazardous environmental stresses, and the exposure to space radiation has been considered as one of the most dangerous factors in Space. For the safety of astronauts, even a low amount of proton exposure in space radiation is necessary to be understood to protect their body systems and maintain the functional balance. However, the endpoint test based on behavioral responses lead to an undetermined consequence in the functional declines by producing the inconsistent results despite of the accumulated proofs in the molecular and the cellular damages by proton. To clarify the systemic link between the proton exposure and the cognitive decline in behaviors, some fundamental behavioral tests, such as rota-rod, open field test, and novel object recognition, were revisited, and the proton-induced alteration was examined by computing behavioral markers in time basis. A portion out of total population (28 SD rats) involved for each test, and the relevant behavioral markers in time were calculated to assess the proton-induced effects in emotion, locomotion and memory. The examination in 3 months of behavioral responses after the different amount of exposure (control, 30cGy-, and 1Gy-exposed animals) identified the emotional and locomotive alterations while few memory-related changes were observed. The computed behavioral markers suggested a quantitative approach to demonstrate the cognitive behavioral effects by a low amount of proton (30cGy) as well as the direct relation between the proton exposure and the cognitive alteration.\u003c/p\u003e","manuscriptTitle":"Proton-induced Behavioral Alterations approved Cognitive Dysfunction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-30 11:37:52","doi":"10.21203/rs.3.rs-3893200/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"26fa027e-4aeb-47c4-a510-f88e1bafcc5d","owner":[],"postedDate":"January 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-02T21:44:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-30 11:37:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3893200","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3893200","identity":"rs-3893200","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}