Cancer treatment induces neuroinflammation and behavioral deficits in mice

Abstract

Introduction: Cancer survivors are increasingly diagnosed with a syndrome of neurocognitive dysfunction termed cancer-related cognitive impairment (CRCI). Chemotherapy and radiation therapy have been implicated in CRCI; however, its underlying pathogenesis remains unclear, hindering effective prevention or treatment. Methods: We used the hairless strain SKH1 (11-12-week-old) and treated the mice with radiation to the right hindlimb, doxorubicin (a chemotherapy agent), concurrent radiation, and doxorubicin, or no treatment (control). Neurocognition was evaluated via standardized behavioral testing following treatment. Mice were subsequently humanely euthanized, and plasma and brains were collected to identify inflammatory changes. Results: Mice treated with radiation, doxorubicin, or both radiation and doxorubicin demonstrated equivalent hippocampal dependent memory deficits and significant increases in activated microglia and astrocytes compared to control mice. Doxorubicin-treated mice had significantly increased plasma IL-6 and failed to gain weight compared to control mice over the study period. Discussion: This study demonstrates that non-brain directed radiation induces both gliosis and neurocognitive deficits. Moreover, this work presents the first characterization of SKH1 mice as a relevant and facile animal model of CRCI. This study provides a platform from which to build further studies to identify potential key targets that contribute to CRCI such that strategies can be developed to mitigate unintended neuropathologic consequences associated with anticancer treatment.

Keywords: SKH1 mice; cancer treatment; cancer-related cognitive impairment; neurobehavior; neuroinflammation.

Figure 1

Experimental design summary. (A) Experimental timeline. (B) Illustration of setup characteristics for each behavior test, including familiarization with objects prior to NLR/NOR testing. OF Test, Open Field Test; NLR, Novel Location Recognition Test; NOR, Novel Object Recognition Test; Y-maze, Spontaneous Alternation y-maze. Created with BioRender.com.

Figure 2

Cancer treatment causes hippocampal dependent memory deficit in mice. (A) Open field test did not detect a difference between groups in the total distance traveled. (B) Open field test did not detect a difference between groups in time spent in the center of the maze. (C) Open field test did not detect a difference between groups in time spent in the periphery of the maze. (D) Open field test did not detect a difference between groups in percentage of time spent in the center of the maze. (E) Novel location testing showed that RT and DOX-RT mice had a significantly lower discrimination ratio than control mice. (F) Novel object recognition test did not detect a difference in discrimination ratio between groups. (G) Spontaneous alternation y-maze testing showed a similar percentage of alternations between groups. Data represent the mean and SEM (n = 12 per group). DOX, doxorubicin group, RT, hindlimb radiation group, DOX-RT, doxorubicin and hindlimb radiation group. Discrimination ratio = (time spent investigating changed object − time spent investigating unchanged object)/(time spent investigating changed object + time spent investigating unchanged object). Spontaneous Alternation percentage (%) = number of spontaneous alternations/(total number of arm entries − 2) × 100. One asterisk (*) representing p-value < 0.05 and two asterisks (**) representing p-value < 0.01.

Figure 3

Cancer therapy causes microgliosis and astrocytosis in the caudal cortex. (A–H) Iba1+ microglia (A,C,E,G) are identified with orange arrows and GFAP+ astrocytes (B,D,F,H) are highlighted with white arrows. Representative images depict microglia and astrocytes from the caudal cortex in control mice (A,B) and mice treated with DOX (C,D), RT (E,F) or DOX-RT (G,H). Images shown are at 200× magnification. (I) Bar graph of Iba1+ microglia across treatment groups. Data represent the mean and SEM evaluated from four to five consecutive images per mouse (n = 12 per group). Mice treated with RT or DOX-RT had significantly more Iba1+ microglia compared to control mice. (J) Bar graph of GFAP+ astrocytes across treatment groups. Data represent the mean and SEM evaluated from four to five consecutive images per mouse (n = 12 per group). Mice treated with DOX-RT had significantly more GFAP+ astrocytes compared to control mice. DOX, doxorubicin group; RT, hindlimb radiation group; DOX-RT, doxorubicin and hindlimb radiation group. One asterisk (*) representing p-value < 0.05 and two asterisks (**) representing p-value < 0.01.

Figure 4

Cancer therapy causes microgliosis and astrocytosis in the cerebellum. (A–H) Iba1+ microglia (A,C,E,G) are identified with orange arrows and GFAP+ astrocytes (B,D,F,H) are shown with white arrows. Representative images depict microglia and astrocytes from the cerebellum in control mice (A,B) and mice treated with DOX (C,D), RT (E,F) or DOX-RT (G,H). Images shown are at 200× magnification. (I) Bar graph of Iba1+ microglia across treatment groups. Data represent the mean and SEM evaluated from four to five consecutive images per mouse (n = 12 per group). Mice treated with RT or DOX-RT had significantly more Iba1+ microglia compared to control mice. (J) Bar graph of GFAP+ astrocytes across treatment groups. Data represent the mean and SEM evaluated from four to five consecutive images per mouse (n = 12 per group). All treated mice had significantly more GFAP+ astrocytes compared to control mice. DOX, doxorubicin group; RT, hindlimb radiation group; DOX-RT, doxorubicin and hindlimb radiation group. One asterisk (*) representing p-value < 0.05 and two asterisks (**) representing p-value < 0.01.

Figure 5

Cancer therapy causes multifocal microgliosis and astrocytosis. (A–E) Bar graphs depicting Iba1+ microglia (top row) and GFAP+ astrocytes (bottom row) are shown from the hippocampus (A), medulla (B), midbrain (C), rostral cortex (D), and striatum (E). Control mice were compared to mice treated with DOX, RT, or DOX-RT. All three treatment groups had significant increases in GFAP+ astrocytes compared to control mice in the hippocampus (A). Mice treated with DOX had more Iba1+ microglia compared to control mice in the medulla (B) and more GFAP+ astrocytes in the midbrain (C). Mice treated with DOX or DOX-RT mice had significantly more Iba1+ microglia compared to control mice in the rostral cortex (D). Mice treated with RT or DOX-RT mice had significantly more Iba1+ microglia compared to control mice in the striatum (E). Data represent the mean and SEM evaluated from four to five consecutive images per mouse (n = 12 per group). DOX, doxorubicin group; RT, hindlimb radiation group; DOX-RT, doxorubicin and hindlimb radiation group. One asterisk (*) representing p-value < 0.05 and two asterisks (**) representing p-value < 0.01.