Effects of dim light at night in C57BL/6J mice on recovery after spinal cord injury

Figures

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  Figure 1.. Experimental groups and timeline of behavioral tests. a. Surgery and treatment groups used in the study. Prior to surgery, all mice (females and males) were housed in light days and dark nights (LD: 12 h light:12 h dark). Mice received sham surgery or moderate 60 kDyn T9 contusion spinal cord injury (SCI). Immediately after surgery, mice were replaced in LD conditions or were newly housed in dim light-at-night (dLAN: light days with dim light-at-night). Mice were housed in these post-surgery lighting conditions for the remainder of the study. b. Schedule of testing for behavioral studies. Locomotor function was assessed repeatedly using the Basso Mouse Scale (BMS). Neuropathic pain-related behaviors were assessed weekly starting at 6–7 dpo using SUDO von Frey test (VF: mechanical allodynia) and Hargreaves test (HG: heat hyperalgesia). Anxiety-like symptoms were tested using the open-field test (OFT) and juvenile social exploration (JSE). Depressive-like symptoms were assessed using sucrose anhedonia (SA) and JSE. All outcomes were assessed prior to and after surgery; see Methods for more details.

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  Figure 2.. Body mass change in mice after SCI and placement in typical light-dark cycles (LD) or light days with dim light-at-night (dLAN). SCI reduced body mass in female and male mice. Lighting condition had no significant effect on body mass changes after SCI (p > 0.05). SCI-LD: n=10; SCI-dLAN: n=8. Plots show mean ± SEM. Hourglass symbol indicates p < 0.001 across timepoints (dpo main effect); * indicates p < 0.001 between pre-surgery and post-SCI body mass at all timepoints for both light treatments; two-way RM ANOVA with Bonferroni post-hoc test.

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  Figure 3.. Effects of dLAN on locomotor recovery after SCI as measured by the Basso Mouse Scale (BMS) score and subscore. A. BMS scores showing SCI-elicited hindlimb locomotor deficits, which recovered over time. B. BMS subscores enabled assessing more detailed recovery of walking patterns after SCI. SCI mice exposed to dLAN had higher BMS scores and subscores at 28 dpo compared to SCI-LD mice. Sham-LD: n=6; Sham-dLAN: n=7; SCI-LD: n=10; SCI-dLAN: n=8. Plots show mean ± SEM. Significant (p < 0.05) surgery (red cross) × dpo (hourglass) interactions were observed via a three-way ANOVA. Timepoints with significantly different SCI vs. Sham results—as determined by Holm-Sidak post-hoc testing—are indicated with a red cross and brackets, while significant SCI-LD vs SCI-dLAN differences are indicated by an asterisk.

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  Figure 4.. Effects of dLAN on contusion SCI-mediated neuropathic pain symptoms. A. SCI induced hypersensitivity to heat from 7–28 dpo as assessed by the Hargreaves test. There was no significant difference in heat sensitivity between LD or dLAN groups. B. Plot of individual withdrawal latencies to heat at 7 dpo to illustrate post-surgery difference of SCI vs Sham animals. C. SCI elicited hypersensitivity to mechanical stimulation as assessed by the von Frey test. SCI-dLAN mice showed a transient increase in mechanical sensitivity at 13 dpo compared to SCI-LD mice. D. Plot of individual SUDO filament thresholds at 13 dpo. Sham-LD: n=6; Shamd-LAN: n=7; SCI-LD: n=10; SCI-dLAN: n=8. Plots show mean ± SEM. Significant (p < 0.05) surgery (red cross) × dpo (hourglass) interactions were observed via a three-way ANOVA. Timepoints with significantly different SCI vs. Sham results—as determined by Holm-Sidak post-hoc testing—are indicated with a red cross, while significant SCI-LD vs SCI-dLAN differences are indicated by an asterisk.

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  Figure 5.. Effects of SCI and dLAN on depressive- and anxiety-like behaviors. A-c. Sucrose anhedonia was used to assess depressive-like behavior prior to surgery and at 14 and 29 dpo. Neither SCI nor dLAN significantly altered sucrose preference. D-f. Open field test: Mice were placed in an open field for 5 minutes to assess locomotion and anxiety-like behavior. D. At 21 dpo, SCI mice traveled less distance than sham mice. E-f. Mice with SCI displayed reduced central tendency at 21 dpo compared to sham mice. G-i. Juvenile social exploration: Mice in an open field were introduced to a novel juvenile; frequency and time of investigation were assessed as measures of anxiety- and depressive-like behavior. H. Mice showed similar behavior at baseline, whereas social exploration was significantly altered by lighting and SCI. i. At 21 dpo, sham-dLAN mice had increased exploration compared to sham-LD mice and SCI-dLAN mice. In addition, SCI-LD mice had increased exploration compared to SCI-dLAN mice. Sham-LD: n=6; Sham-dLAN: n=7; SCI-LD: n=9–10; SCI-dLAN: n=9. Plots show mean ± SEM. Significant (p < 0.05) main or interaction (indicated by “x”) effects from two or three-way ANOVA tests are indicated…

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  Figure 6.. dLAN did not significantly alter post-SCI lesion size and tissue sparing in C57BL/6J mice. 10 μm spinal cord cryosections from 35 dpo mice were imaged at regular 0.2 mm intervals up to −1 mm rostral and +1 mm caudal of the lesion epicenter. A. Representative epicenter spinal cord cross-sections from SCI mice exposed to LD or dLAN. Sections were immunostained with anti-GFAP and anti-Iba1 to detect reactive astrocytes and microglia/macrophages respectively, along with DAPI to counterstain nuclei. Lesion area was determined by manually tracing the border based on anti-GFAP staining. B. dLAN after SCI had no significant effect on lesion size nor tissue sparing. Spared tissue was determined by subtracting the lesion area from the total area of the spinal cord section—this is expressed as the percentage of spared tissue relative to the total area. Lighting condition had no significant effect on either lesion area or tissue sparing (p = 0.61 and 0.33 respectively) based on two-way repeated measures ANOVA. SCI-LD: n=9; SCI-dLAN: n=7. Plots show mean ± SEM. Scale bars represent 500 μm.