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. 2021 Mar 15:1755:147275.
doi: 10.1016/j.brainres.2020.147275. Epub 2021 Jan 7.

Secondary-blast injury in rodents produces cognitive sequelae and distinct motor recovery trajectories

Jasmine Gamboa  1 , Jessica Horvath  1 , Amanda Simon  1 , Md Safiqul Islam  1 , Sijia Gao  1 , Dror Perk  1 , Amy Thoman  1 , Diany Paola Calderon  2
Affiliations

Secondary-blast injury in rodents produces cognitive sequelae and distinct motor recovery trajectories

Jasmine Gamboa et al. Brain Res. .
No abstract available

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Conflict of interest statement

Conflict of interest:

The authors declare no competing financial or non-financial interests as defined by Elsevier’s guidance.

Figures

Figure 1.
Figure 1.. Development of a secondary blast injury model and timeline of experimental design.
(A) Close up of head holder and lateral view of the impacted area (mesocircuit). The photograph shows a micro-switch sensor that limits the penetration of the piston (which represents shrapnel associated in SBI) in the region of interest. The animal is exposed to isoflurane via an anesthetic mask as depicted. (B) Gross pathology showing the injured area on a perfused brain after right SBI from a lateral(B) and dorsal view (C) at 4 hours and 21 days post-SBI. Pressure-dependent apparatus for producing SBI including the components used to align the head and the modified blast gun aligned with the target area using a motor stepper controlled by an Arduino (D) Experimental timeline including all behavioral assessments conducted in male and female mice. The brains were perfused following behavioral experiments. Scale bar: 3.5 mm
Figure 2.
Figure 2.. Blast injured male mice exhibit memory impairment during the learning phase of the Morris water maze task.
(A) Swim duration of each animal per trial and day during the learning phase (4 days). SBI data is shown in red, and control (CTRL) in black. If animals reached the platform before the established time (90s), we identified the trace with a closed circle. However, if the animal could not reach the platform within the 90s, we identified it with an open circle (censored time) (B) Probability of reaching the platform as a function of time (4 days) for SBI (red) and control (black) mice applying predictions from the accelerated failure time (AFT) time-to-event model. See table 4 for p-values. The interval (gray) represents the standard error. See methods for data analysis in section 2.2 & 4.7.3
Figure 3.
Figure 3.. Blast injured males are impaired in extinguishing previously learned behavior.
(A) The schematic displays the location of the platform in Quadrant 1 during the learning days. After the first four days of learning, the platform was moved to Quadrant 3 for the probe days. (B) SBI male(n=54) average latencies (mean ± S.E.M) to find the platform compared to controls(n=28) on probe days. A two-way repeated measures ANOVA analysis conducted on the probe phase latencies reveal a significant effect between groups (F(1,268)=26.03, p<0.001). SBI males exhibited significantly longer latencies than controls on days 2 and 4 of the probe phase (p<0.05, Bonferroni-adjusted T-test). (C) During the probe phase, SBI males spent more time on average (mean ± S.E.M) in Quadrant 1 (Q1) and less time in Quadrant 3 (Q3), while controls spent more time on average in Q3 and less time in Q1 as they learned the new platform location (p<0.05, Bonferroni-adjusted T-test). Our analysis of the percentage of time the groups spent in each quadrant revealed that the quadrant*group interaction was significant (F(3,4480)=7.47, p<0.001 ; three-way ANOVA). No changes in other quadrants were observed. (*p<0.05, **p<0.01, ***p<0.001).
Figure 4.
Figure 4.. SBI and control rotarod (RR) performances under two different motor learning paradigms.
(A) Evaluation of trajectories of control group (n=28) by applying principal component analysis (PCA). We calculated the Euclidian distance between controls and the centroid. The integer that enclosed the majority (>90%) of controls was considered as the radius of the gray circle displayed in the figure. (B) Corresponding trajectories per animal of the control group between day 0-3 (C) Data distribution of SBI trajectories along the principal component 1 (PC1) and classification. If the distance was less than the radius of control PCs in our SBI group, we identified the cluster as standard (SBI-S;n=21). We defined low-performers (SBI-L;n=5) if the 1st PC was negative and high-performers (SBI-H;n=19) if the 1st PC was positive.(D) Corresponding classified trajectories per animal of the SBI group between day 0-3 (E) One week after the injury, SBI-mice that were high performers (SBI-H mice) ran longer average durations (mean ± S.E.M) on the RR than low-performers (SBI-L),standard and control mice. A two-way repeated measures ANOVA conducted on the run times revealed a significant between-group effect (F(3, 322)=87.20, p<0.001) and significant within-group effects in the group*day interaction (F(23.35, 2505.76)=1.66, p<0.05; Huynh-Feldt corrected). SBI-H mice had significantly longer run times compared to the SBI-L, (F(1,97)=73.40, p<0.001), SBI-S (F(1,175)=95.96, p<0.001), and control groups (n=28; F(1,228)=140.87, p<0.001). Additionally, SBI-L mice had significantly lower run times than control (F(1,147)=68.69, p<0.001), SBI-S (F(1,94)=88.83, p<0.001), and SBI-H mice (F(1,97)=73.40, p<0.001). There were no significant differences found between control and SBI-S mice (F(1,225)=0.56, p=0.45). (F) No significant differences were observed during the pretraining days (Days 6-8) between groups. SBI-H mice (n=9) significantly improved their RR performance after the injury (Day 17). In contrast, RR performance by SBI-L mice (n=5) worsened following injury. No change in motor learning behavior was observed among control mice (n=20) and SBI-S group(n=10). A two-way repeated measures ANOVA conducted on run times revealed a significant between-group effect (F(3,126)=12.68, p<0.001) and significant within-group effects of day and the cluster per day interaction (F(11.56,1456.43)=11.55, p<0.001 and F(F(34.67,1456.43)=7.86, p<0.001 respectively; Greenhouse-Geisser corrected). (*p<0.05, **p<0.01, ***p<0.001).
Figure 5.
Figure 5.. Absence of anxiety-like behaviors in SBI mice.
(A) SBI males (n=14) performed similarly to controls (n=5) in the Open Field Test (OFT) when assessing average distance traveled, speed (p=0.97 and p=0.96 respectively, one-way ANOVA), and time spent in center and peripheral zones within the chamber (p=0.46, two-way ANOVA). The circles represent averaged trial scores for each subject. (B) SBI females (n=8) performed similarly to controls (n=8) in distance traveled, speed (p=0.62 and p=0.79 respectively, one-way ANOVA), and time spent in each zone (p=0.51, two-way ANOVA). NS. Non-significant statistical differences.
Figure 6.
Figure 6.. Quantification of neuron degeneration in SBI mice.
Representative brain section from a control(A) and SBI (C) mice displaying Fluoro-jade staining in the midbrain area (Bregma: −3.95 mm). Scale bar= 500 μm. Magnification of the region of interest (green rectangle) depicting the superior colliculus in control (B) and SBI mice(D). Scale bar=100 μm. (E) shows automated segmentation of cell counts represented as mean ± S.E.M where cell densities for SBI mice(n=6) were significantly higher than controls (n=3; one-tailed Wilcoxon test) *p<0.05, **p<0.01. PAG periaqueductal grey, PPN, pedunculopontine tegmentum, CA1 and CA3 area of the hippocampus, SUB, subiculum, SC, superior colliculus, and PG, pontine gray.
See this image and copyright information in PMC

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