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. 2022 Sep 16;17(9):e0274007.
doi: 10.1371/journal.pone.0274007. eCollection 2022.

Cognitive and behavioral effects of whole brain conventional or high dose rate (FLASH) proton irradiation in a neonatal Sprague Dawley rat model

Michael T Williams  1   2   3 , Chiho Sugimoto  2 , Samantha L Regan  1   2 , Emily M Pitzer  1   2 , Adam L Fritz  2 , Mathieu Sertorio  3   4 , Anthony E Mascia  3   4 , Ralph E Vatner  3   4 , John P Perentesis  1   3   5 , Charles V Vorhees  1   2   3
Affiliations

Cognitive and behavioral effects of whole brain conventional or high dose rate (FLASH) proton irradiation in a neonatal Sprague Dawley rat model

Michael T Williams et al. PLoS One. .

Abstract

Recent studies suggest that ultra-high dose rates of proton radiation (>40 Gy/s; FLASH) confer less toxicity to exposed healthy tissue and reduce cognitive decline compared with conventional radiation dose rates (~1 Gy/s), but further preclinical data are required to demonstrate this sparing effect. In this study, postnatal day 11 (P11) rats were treated with whole brain irradiation with protons at a total dose of 0, 5, or 8 Gy, comparing a conventional dose rate of 1 Gy/s vs. a FLASH dose rate of 100 Gy/s. Beginning on P64, rats were tested for locomotor activity, acoustic and tactile startle responses (ASR, TSR) with or without prepulses, novel object recognition (NOR; 4-object version), striatal dependent egocentric learning ([configuration A] Cincinnati water maze (CWM-A)), prefrontal dependent working memory (radial water maze (RWM)), hippocampal dependent spatial learning (Morris water maze (MWM)), amygdala dependent conditioned freezing, and the mirror image CWM [configuration B (CWM-B)]. All groups had deficits in the CWM-A procedure. Weight reductions, decreased center ambulation in the open-field, increased latency on day-1 of RWM, and deficits in CWM-B were observed in all irradiated groups, except the 5 Gy FLASH group. ASR and TSR were reduced in the 8 Gy FLASH group and day-2 latencies in the RWM were increased in the FLASH groups compared with controls. There were no effects on prepulse trials of ASR or TSR, NOR, MWM, or conditioned freezing. The results suggest striatal and prefrontal cortex are sensitive regions at P11 to proton irradiation, with reduced toxicity from FLASH at 5 Gy.

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Conflict of interest statement

These experiments were funded by Varian, a Siemens Healthineers company that granted the authors intellectual freedom to publish the data. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Experimental timeline.
The experimental timeline in postnatal days. Abbreviations: ASR- acoustic startle response; TSR- tactile startle response; PPI- prepulse inhibition; NOR- novel object recognition; RWM- radial water maze; CF- conditioned freezing; CWM- Cincinnati water maze. Created with BioRender.com.
Fig 2
Fig 2. Body weights [g (mean ± SEM)] of proton irradiated and control rats.
A. The overall effect on body weights by exposure group, averaged across sex and week. B. Body weights of females by weeks. C. Body weights of males by weeks. While there was an exposure × sex interaction, both females and males dosed with 8 Gy were lighter than controls. See text for a summary of the exposure × age interaction. ***p < 0.001 compared with controls. N = 14-19/sex/exposure at each week. Due to scheduling errors some of the body weights were missed on some weeks.
Fig 3
Fig 3. Locomotor activity [beam breaks (mean ± SEM)] of proton irradiated and control rats.
A. Effects on ambulation by exposure, averaged across sex and test interval (time). No differences in irradiated groups compared with controls. B. Ambulation by test interval with males and females combined. C. Effects on center ambulation by exposure, averaged across sex and time. All irradiated groups with the exception of the 5-FLASH group had reduced center ambulation compared with controls. D. Center ambulation by test interval with males and females combined. *p < 0.05 compared with controls. N = 19/sex/exposure except control male = 17 and 5-FLASH male and female = 18.
Fig 4
Fig 4. Acoustic or tactile startle responses [Vmax (mean ± SEM)] with or without acoustic or light prepulses.
A. The effect on acoustic startle response by exposure, averaged across sex and days. The 8-FLASH group had reduced acoustic startle compared with controls. B. The effect on tactile startle response by exposure, averaged across sex and days. The 8-FLASH group had reduced tactile startle compared with controls. For panels A & B, N = 19/sex/exposure except Control and 5-FLASH males = 18/group. C. The effect of acoustic prepulses prior to an acoustic pulse by exposure averaged across sex. There were no differences between controls and irradiated groups. D. The effect of acoustic prepulses prior to a tactile pulse by exposure averaged across sex. There were no differences between controls and irradiated groups. For panels C & D, N = 19/sex/exposure except 5-FLASH females and males = 18 and Control males = 17. Two rats were found outside of the holder and their data were not used. E. The effect of light prepulses prior to an acoustic pulse by exposure averaged across sex. There were no differences between controls and irradiated groups. F. The effect of light prepulses prior to a tactile pulse by exposure averaged across sex. There were no differences between controls and irradiated groups. For panels E & F, N = 16/sex/exposure except 5-FLASH males = 15 and Control males = 14. The discrepancy in numbers was the result of the computer program failing to save data from rats that were run in the test. *p < 0.05.
Fig 5
Fig 5. The Cincinnati water maze latency [s (mean ± SEM)] and number of errors (mean ± SEM) of proton irradiated and control rats.
A. The overall effect on latency where irradiated groups took longer to locate the platform compared with controls. B. The learning curves for latency. There was an interaction of exposure x day that is described in the results. Differences between the controls and irradiated groups began after day 5 and from day 10 to 18 all groups except the 5-FLASH group took longer to locate the platform. C. The overall effect on errors where irradiated groups had more errors to locate the platform. D. The learning curves for errors. Similar to latency there was an exposure x day interaction that is described in the results. N = 19/sex/exposure except control male and 5-FLASH male = 18. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with controls.
Fig 6
Fig 6. The radial water maze latency [s (mean ± SEM)] and number of errors (mean ± SEM) of proton irradiated and control rats on Day 1 and Day 2.
A. The overall effect on latency in the radial water maze on Day 1. With the exception of the 5-FLASH group, the irradiated groups took longer to locate the platform. B. The overall effect on mean errors per trial in the radial water maze on Day 1. The 5-Conv and 8-Conv group made more errors compared with controls, while there was no difference for the FLASH groups and controls. N = 19/sex/exposure except control male and 5-FLASH male = 18. C. The overall effect on latency in the radial water maze on Day 2. The 5-FLASH and 8-FLASH groups took longer to locate the platform compared with controls. D. The overall effect on mean errors in the radial water maze on Day 2. There were no differences between irradiated groups and controls. There was an interaction of exposure × sex × trial where males in the 5-FLASH and 8-Conv groups made more errors on trial-7 only (not shown). N = 19/sex/exposure except control male, 5-FLASH male, and 8-Conv females = 18. One 8-Conv female was inadvertently missed. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with controls.
Fig 7
Fig 7
The Morris water maze latency [s (mean ± SEM)], path efficiency (mean ± SEM), and average distance from the platform on probe trials [m (mean ± SEM)] for the acquisition (A, B, C), reversal (D, E, F), and shift (G, H, I) phases and latency [s (mean ± SEM)] during the cued (J) phase of proton irradiated and control rats. There were no overall differences between the irradiated groups and controls on any measure in the Morris water maze. There was a significant interaction of exposure × day for acquisition latency where the 5-Conv and 8-Conv groups took longer to locate the platform on days 2 and 3 compared with the controls, *p < 0.05. No other interactions involving exposure were found. N = 19/sex/exposure except control male and 5-FLASH male = 18.
Fig 8
Fig 8. Conditioned freezing [beam interruptions (mean ± SEM)] for proton irradiated and control rats.
A. Day 1 habituation phase and conditioning phase; there were no differences between the irradiated groups and controls. B. Day 2 contextual learning phase; there were no differences between irradiated groups and controls. C. Day 3 extinction phase for the tone on trials; no differences between groups. D. Day 4 reinstatement phase; no differences were noted. N = 16-19/exposure/sex. Several rats escaped the test arena during the conditioning phase and their data were not used.
Fig 9
Fig 9. The Cincinnati water maze mirror version latency [s (mean ± SEM)] and number of errors (mean ± SEM) of proton irradiated and control rats.
A. The overall effect on latency where irradiated groups with the exception of the 5-FLASH group took longer to locate the platform compared with controls. B. The learning curves for latency. All exposure groups had decreased latencies over days. C. The overall effect on errors where irradiated groups with the exception of the 5-FLASH group had more errors to locate the platform compared with controls. D. The learning curves for errors. Errors decreased over days for all groups. N = 19/sex/exposure except control male and 5-FLASH male = 18. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with controls.
Fig 10
Fig 10
Physiological measures in irradiated and control rats in the neostriatum (A-E) and hippocampus (F). A. For TH there was no overall difference between irradiated groups and controls, but there was an exposure × sex interaction. There were no differences in males whereas the FLASH females, regardless of dose, had increased TH levels compared with control females. B. For DAT the 5-Conv group had increased levels of DAT compared with the controls. C. For DRD1 there was an increase in receptor levels for the groups that received 8 Gy, regardless of dose rate, compared with controls. D. There was an exposure × sex interaction. There were no differences in males, whereas the 8-FLASH females had increased DRD1 levels compared with control females. ELISA was used for Panels A-D, N = 8/sex/exposure. E. There were no differences in DRD2 in the neostriatum. F. There were no differences for NMDA-R1 in the hippocampus. Westerns were used for panels E-F, N = 6-7/sex/exposure.
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