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. 2020 May;193(5):407-424.
doi: 10.1667/RR15540.1. Epub 2020 Mar 5.

Neurological Impairments in Mice Subjected to Irradiation and Chemotherapy

Deblina Dey  1 , Vipan K Parihar  1 , Gergely G Szabo  2 , Peter M Klein  2 , Jenny Tran  1 , Jonathan Moayyad  1 , Faizy Ahmed  3 , Quynh-Anh Nguyen  2 , Alexandria Murry  1 , David Merriott  1 , Brandon Nguyen  1 , Jodi Goldman  1 , Maria C Angulo  1 , Daniele Piomelli  3 , Ivan Soltesz  4 , Janet E Baulch  1 , Charles L Limoli  1
Affiliations

Neurological Impairments in Mice Subjected to Irradiation and Chemotherapy

Deblina Dey et al. Radiat Res. 2020 May.

Abstract

Radiotherapy, surgery and the chemotherapeutic agent temozolomide (TMZ) are frontline treatments for glioblastoma multiforme (GBM). However beneficial, GBM treatments nevertheless cause anxiety or depression in nearly 50% of patients. To further understand the basis of these neurological complications, we investigated the effects of combined radiotherapy and TMZ chemotherapy (combined treatment) on neurological impairments using a mouse model. Five weeks after combined treatment, mice displayed anxiety-like behaviors, and at 15 weeks both anxiety- and depression-like behaviors were observed. Relevant to the known roles of the serotonin axis in mood disorders, we found that 5HT1A serotonin receptor levels were decreased by ∼50% in the hippocampus at both early and late time points, and a 37% decrease in serotonin levels was observed at 15 weeks postirradiation. Furthermore, chronic treatment with the selective serotonin reuptake inhibitor fluoxetine was sufficient for reversing combined treatment-induced depression-like behaviors. Combined treatment also elicited a transient early increase in activated microglia in the hippocampus, suggesting therapy-induced neuroinflammation that subsided by 15 weeks. Together, the results of this study suggest that interventions targeting the serotonin axis may help ameliorate certain neurological side effects associated with the clinical management of GBM to improve the overall quality of life for cancer patients.

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Figures

FIG. 1.
FIG. 1.
Combined treatment promotes anxiety-like tendencies in an open field. Panel A: Experimental design. Six-month-old male C57BL/6J mice received three X-ray doses of 8.67 Gy in week 1 interspersed with concomitant doses of TMZ administered intraperitoneally (i.p., 25 mg/kg). Mice then received nine adjuvant doses (66.7 mg/kg) of TMZ (i.p.) in the subsequent 3 weeks (3 alternating days/week). The first cohort of mice was subjected to behavioral testing at 5 weeks postirradiation and the second at 15 weeks postirradiation followed by immunohistochemistry and HPLC studies for both cohorts and electrophysiology for the 15-week cohort. These time points were specifically counted from the final day of irradiation. Panel B: Representative movement tracking of control and combined treatment mice in the 5-min open field test at 5 weeks postirradiation. Panel C: Combined treatment mice traveled significantly less distance than control mice (408 ± 123 cm and 1,004 ± 120 cm, respectively) and spent significantly less time in the central zone compared to the control animals (10.2 ± 2.0 s and 23.6 ± 4.0 s, respectively). Panel D: Representative movement tracking of control and combined treatment mice in the 5-min open field test at 15 weeks postirradiation. Panel E: Combined treatment mice travelled significantly less distance than control mice (817 ± 130 cm and 1,487 ± 80 cm, respectively) and spent significantly less time in the central zone compared to the control animals (6.9 ± 2.0 s and 13.6 ± 3.0 s, respectively). For the 5-week cohorts, n = 10 for the control and n = 9 for the combined treatment group. For the 15-week cohorts, n = 15 for the control and n = 14 for the combined treatment group. Data are presented as mean ± SEM. P values are derived using the Mann-Whitney U test. *P < 0.05, **P < 0.01, ***P < 0.001.
FIG. 2.
FIG. 2.
Combined treatment induces anxiety- and depression-like behavior. Panel A: At 5 weeks postirradiation combined treatment mice spent a similar percentage of time in the open arms of the EPM compared to the control mice (19.3 ± 3.8 and 23 ± 2.0, respectively), but differed significantly in the percentage of entries into the open arms (47 ± 0.6 and 49 ± 0.2, respectively). Panel B: At 15 weeks postirradiation, the percentage of time spent in the open arms of the EPM by combined treatment mice was less than that of control mice (6.3 ± 1.5 and 15.4 ± 2.0, respectively), but combined treatment mice made more entries into the open arms compared to controls (81.2 ± 4.0 and 68.6 ± 2.8, respectively). Panel C: At 5 weeks postirradiation no significant difference in the time spent in the light compartment of the light-dark box arena was found between combined treatment and control mice (45.2 ± 8.7 and 106.3 ± 23.1, respectively). However, combined treatment mice had fewer numbers of transitions between compartments compared to controls (10 ± 2.0 and 22 ± 4.0, respectively). Panel D: At 15 weeks postirradiation, combined treatment and control mice spent similar times in the light compartment of the light-dark box (79.7 ± 18.7 and 104.4 ± 20.5, respectively). However, combined treatment mice had fewer numbers of transitions between compartments compared to controls (13 ± 2.0 and 21 ± 3.0, respectively). Panel E: At 5 weeks postirradiation on the forced swim test the combined treatment and control mice spent similar amounts of time floating (118.2 ± 14.0 s and 135.7 ± 9.0 s, respectively), but at the late time point the combined treatment mice spent significantly more time floating than did the controls (132 ± 14.1 s and 83 ± 12.7 s, respectively). For the EPM and LDB 5-week cohorts, n = 10 for the control group and n = 9 for the combined treatment group; for the 15-week cohort, n = 15 for the control group and n = 14 for the combined treatment group. For the FST 5-week cohorts, n = 9 each for the control and combined treatment groups; and for the 15-week cohort, n = 6 each for the control and combined treatment groups. Data are presented as mean ± SEM. P values are derived from the Mann-Whitney U test. *P < 0.05, **P < 0.01.
FIG. 3.
FIG. 3.
Individual treatment with radiation or TMZ does not cause anxiety. Panels A–C: Representative movement tracking of 0 Gy (control), 26 Gy (radiation alone) and TMZ-only treated mice, respectively, in the 5min open field test. Panel D: No significant difference in distance travelled was observed in individually treated groups of mice (536 ± 120 cm, 707 ± 88 cm and 351 ± 90 cm, for control, 26 Gy and TMZ, respectively). Panel E: Similarly, no significant difference in time spent in the center zone was observed in individually treated groups of mice (7.8 ± 2.6 s, 3.7 ± 0.6 s and 3.4 ± 1.4, for control, 26 Gy and TMZ, respectively). Panel F: The EPM test also revealed no significant difference in the percentage of time spent in open arms of the maze between the three groups (23 ± 3, 23 ± 3 and 18 ± 3 for control, 26 Gy and TMZ, respectively); and (panel G) the number of the entries into the open arms was also similar among all groups (49 ± 0.3, 49 ± 0.4 and 43 ± 6 for control, 26 Gy and TMZ, respectively). Panel H: LDB testing similarly showed no differences in the time spent in the light compartment among groups (63 ± 14, 62 ± 10 and 74 ± 26 for control, 26 Gy and TMZ, respectively) and (panel I) no difference in the number of transitions (10 ± 2, 14 ± 2 and 11 ± 3 for control, 26 Gy and TMZ, respectively). Panel J: In the FST, the irradiated mice exhibited more time floating than either control or TMZ-treated mice (165 ± 5, 188 ± 5 and 175 ± 8 for control, 26 Gy and TMZ, respectively). For these studies, the control n = 12 for all tasks; the 26 Gy cohort n = 14 for panels A–I and n = 13 for panel J; and the TMZ cohort n = 7 for panels A–G, n = 6 for panels H–I and n = 5 for panel J. Data are presented as mean ± SEM. P values are derived from one-way ANOVA. **P < 0.01
FIG. 4.
FIG. 4.
Effects of combined treatment on cognitive impairments using the MWM at 15 weeks postirradiation. Panel A: The swimming velocity (cm/s) was similar between control and combined treatment mice; and (panel B) no change was found in learning between the combined treatment and control mice, as measured by latency to locate the platform. Panel C: As measured by dwell time, control mice exhibited a clear preference for the original platform quadrant, which was greater than the time spent in the right and opposite quadrant during the memory retrieval test (right 13.3 ± 1.5 s; opposite 12.3 ± 0.9 s; left 14.5 ± 1.4 s; platform 18.5 ± 1.7 s); however, (panel D), combined treatment mice exhibited no quadrant preference during the memory retrieval task (right 13.3 ± 1.8 s; opposite 14.8 ± 1.1 s; left 14.9 ± 1.3 s, platform 16.3 ± 1.7 s). Control n = 13 and combined treatment n = 9. Data are presented as mean ± SEM. P values are derived from (panels A and B) two-way RM ANOVA with Bonferonni’s multiple comparisons. Panels C and D: Paired Student’s t test was used; *P < 0.05. Panels E and F: Wilcoxon matched-pairs signed rank test was used.
FIG. 5.
FIG. 5.
Electrophysiological properties of CA1 pyramidal neurons are not altered after combined treatment. All data are from whole-cell recordings of CA1 pyramidal neurons from the superficial layer of the ventral hippocampus. Panel A: Resting membrane potential was unchanged between combined treatment and control mice. Panel B: Representative examples of responses to a range of brief current injections in control and combined treatment neurons. There was no alteration in the (panel C) input resistance, (panel D) sag during a −100 pA hyperpolarizing current injection, (panel E) or rheobase current required to evoke an action potential between the treatment groups. Panel F: Representative examples of recordings containing spontaneous EPSCs from control and combined treatment neurons. Panel G: The frequency of sEPSCs was equivalent between control and combined treatment neurons. Panel H: Examples of EPSCs in representative control and combined treatmnet neurons. Light lines show individual sEPSCs, while the darker line displays the average sEPSC during a 200-s recording from that neuron. Neither (panel I) sEPSC amplitude nor (panel J) the sEPSC decay time-constant was shifted between groups. Control n = 16 cells and combined treatment n = 25 cells for all plots showing grouped data.
FIG. 6.
FIG. 6.
Microglial activation is acutely elevated by combined treatment. Representative images of CD68+ activated microglia immunohistochemistry in the CA1 (panels A and B) and CA3 (panels C and D) regions of the hippocampus of control and combined treatment mice at 5 weeks postirradiation. CT = combined treatment. Panel E: Increased numbers of activated microglia were found at 5 weeks postirradiation in the CA1 region of the hippocampus of combined treatment mice compared to controls (4854 ± 413 control and 1690 ± 407 combined treatment, CD68+ cells) and (panel F) in the CA3 region of combined treatment mice compared to controls (3526 ± 254 control and 681.8 ± 199 combined treatment, CD68+ cells). Relative to controls, no change in microglial activation was observed at 15 weeks postirradiation in either the CA1 (panel G; 20,060 ± 197 control and 22,050 ± 274 combined treatment, CD68+ cells) or CA3 (panel H) region of the hippocampus (16,540 ± 411 control and 15,770 ± 167 combined treatment, CD68+ cells). Black indicates the CD68+ cells and red indicates the nuclear fast red counterstain; Scale bar = 30 μm. The number of CD68+ cells in each combined treatment group is normalized to the number of CD68+ cells in the control group and presented as mean ± SEM for n = 4 mice/group. P values are derived from the Mann-Whitney U test. *P < 0.05.
FIG. 7.
FIG. 7.
Combined treatment alters hippocampal serotonin receptor 5HT1A receptor (5HT1AR) levels. Panels A and B: Representative images of 5HT1AR immunohistochemistry in the CA1 region of the hippocampus in control and combined treatment mice at 5 weeks postirradiation, respectively. Panel C: Combined treatment mice had reduced numbers of 5HT1AR puncta per μm3 × 100 in the CA1 region of the hippocampus compared to control mice at 5 weeks postirradiation (0.92 ± 0.1 and 2.7 ± 0.1, respectively). Panels D and E: Representative images of 5HT1A receptor immunohistochemistry in the CA3 region of the hippocampus in control and combined treatment mice at 5 weeks postirradiation, respectively. Panel F: At 5 weeks postirradiation, fewer 5HT1AR puncta were also observed in the CA3 region of the hippocampus of combined treatment compared to control mice (0.92 ± 0.1 and 2.02 ± 0.2, respectively). Panels G and H: Representative images of 5HT1A receptor immunohistochemistry in the CA1 region of the hippocampus in control and combined treatment mice at 15 weeks postirradiation, respectively, at which time (panel I), combined treatment mice had reduced numbers of 5HT1AR puncta in the CA1 region of the hippocampus compared to control mice (1.5 ± 0.1 and 3.3 ± 0.4, respectively). Panels J and K: Representative images of 5HT1A receptor immunohistochemistry in the CA3 region of the hippocampus in control and combined treatment mice, respectively, at 15 weeks postirradiation, at which time (panel L) levels of 5HT1AR in the CA3 region of the hippocampus of combined treatment mice were indistinguishable from that of controls (2.5 ± 0.4 and 3.2 ± 0.6, respectively). Red indicates 5HT1AR; blue indicates DAPI counterstain. Scale bar = 20 μm. SP = stratum pyramidale, SR = stratum radiatum and SLM = stratum lacunosum moleculare. Data are presented as mean ± SEM for n = 4 mice/group. P values are derived using Mann-Whitney U test. **P < 0.01, ***P < 0.001.
FIG. 8.
FIG. 8.
Hippocampal serotonin levels are reduced after combined treatment, but not after individual therapies. Panel A: The levels of 5-HT were similar in combined treatment and control mice at 5 weeks postirradiation. Panel B: However, at 15 weeks postirradiation combined treatment mice had lower levels of 5-HT compared to controls. Panel C: Compared to controls, mice treated with radiation alone (26 Gy) or with TMZ alone showed no evidence of decreased hippocampal levels of 5-HT at 15 weeks postirradiation. Levels of 5-HT are normalized to that of the control. For the combined treatment study, data are presented as mean ± SEM, n = 6–8 mice/group, and P values are derived using Mann-Whitney U test. *P < 0.05. For the individual therapy study, data are presented as mean ± SEM n = 4–6 mice/group and P values are derived using one-way ANOVA.
FIG. 9.
FIG. 9.
Fluoxetine alleviates combined treatment-induced depression-like behavior. Panel A: Six-month-old male mice received combined treatment and were then allowed to recover for one week before beginning fluoxetine (FLX) treatment (25 mg/kg/day). Mice remained on FLX until 15 weeks postirradiation at which time FST was conducted. Panel B: Mice receiving combined treatment with FLX spent significantly less time floating than mice given combined treatment alone (66.5 ± 5.1 s and 101.5 ± 14.0 s, respectively). Similarly, combined treatment mice spent significantly more time floating than controls (101.5 ± 14.0 s and 70.0 ± 5.8 s, respectively). No difference in floating time was found between control mice and those receiving combined treatment with FLX (P = 0.8). For these studies, n = 8 for the controls, n = 7 for the combined treatment group and n = 6 for the combined treatment with FLX group. Data are presented as mean ± SEM. P values are derived using one-way ANOVA. *P < 0.05.
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