Effects of thioTEPA chemotherapy on cognition and motor coordination

Abstract

Cancer survivorship has increased greatly as therapies have become more advanced and effective. Thus, we must now focus on improving the quality of life of patients after treatment. After chemotherapy, many patients experience chemotherapy-induced cognitive decline, indicating a need to investigate pathologies associated with this condition. In this study, we addressed cognitive impairment after thioTEPA treatment by assessing behavior and assaying cytokine production and the structure of dendrites in the hippocampus. Male mice were given three intraperitoneal injections of thioTEPA. Five weeks later, the mice underwent behavior testing, and brains were collected for Golgi staining and cytokine analysis. Behavior tests included y-maze and Morris water maze and licking behavioral task. Cytokines measured include: IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-10, IL-12p70, MCP-1, TNF-α, GMCSF, and RANTES. We observed decreased memory retention in behavioral tasks. Also, dendritic arborization and length were decreased after chemotherapy treatment. Finally, thioTEPA decreased cytokine production in animals treated with chemotherapy, compared to saline-treated controls. Here, we used a mouse model to correlate the decreases in dendritic complexity and inflammatory cytokine production with cognitive impairment after chemotherapy.

Figure 1:. TH-treatment significantly decreased the amount of time mice spent in the novel arm of the Y-maze.

Saline-treated mice spent significantly more time exploring the novel arm compared to the start or familiar arms. ThioTEPA-treated mice spent their time randomly exploring the familiar, start, and novel arms. Each bar represents the mean of 8–10 mice; error bars are the SEM. ****P < 0.0001

Figure 2:. Spatial memory retention during probe trials on days 3–5 of Morris water-maze testing.

(a, b, and c) Saline probe trials; saline-treated animals spent significantly more time in the target quadrant than other quadrants. (d, e, and f) ThioTEPA-treated mice spent the same amount of time in each of the quadrants in probe trials. Each bar represents the mean of 8–10 mice; error bars are the SEM. * P < 0.05; ** P < 0.01

Figure 3:. TH- treatment significantly decreased dendritic complexity throughout the DG region of the hippocampus.

a) Dendritic length measured by Sholl analysis revealed a decrease in arborization particularly evident at distances between 50-150 μm from the soma b) TH-treatment significantly decreased complexity. Average ± SEM (n = 5); * P < 0.05; ** P < 0.01; ‡ P < 0.001.

Figure 4:. Sholl analyses of CA1 pyramidal neurons

(a) Dendritic length measured by Sholl analysis in CA1 apical revealed a decrease in arborization at 40–130 μm from the soma after exposure to ThioTEPA. (b) ThioTEPA exposure greatly decreased overall dendrite complexity. (c) In the CA1 basal, mice exposed to ThioTEPA showed a decrease in arborization at 30–130 μm from the soma. (d) ThioTEPA exposure greatly decreased overall dendrite complexity in CA1 basal area. Average ± SEM (n = 5); * P < 0.05; **** P < 0.0001; ‡ P < 0.001.

Figure 5:. Analysis of dendritic complexity in CA3 apical region of thioTEPA-treated mice versus saline-treated animals.

(a) Dendritic length measured by Sholl analysis in CA3 apical revealed a decrease in arborization at 60–160 μm from the soma that was particularly evident after exposure to ThioTEPA. (b) ThioTEPA exposure greatly decreased overall dendritic complexity. (c) In the CA3 basal, mice exposed to ThioTEPA showed a decrease in arborization at 60–170 μm from the soma. (d) ThioTEPA exposure greatly decreased overall dendrite complexity in CA3 basal area. (e-f) Representative tracings of CA3 neurons superimposed over concentric rings (10 μM) used for Sholl analyseAverage ± SEM (n = 5); * P < 0.05; ‡ P < 0.001.

Figure 5:. Analysis of dendritic complexity in CA3 apical region of thioTEPA-treated mice versus saline-treated animals.

(a) Dendritic length measured by Sholl analysis in CA3 apical revealed a decrease in arborization at 60–160 μm from the soma that was particularly evident after exposure to ThioTEPA. (b) ThioTEPA exposure greatly decreased overall dendritic complexity. (c) In the CA3 basal, mice exposed to ThioTEPA showed a decrease in arborization at 60–170 μm from the soma. (d) ThioTEPA exposure greatly decreased overall dendrite complexity in CA3 basal area. (e-f) Representative tracings of CA3 neurons superimposed over concentric rings (10 μM) used for Sholl analyseAverage ± SEM (n = 5); * P < 0.05; ‡ P < 0.001.

Figure 6:. ThioTEPA treatment significantly decreased the amount of IL-2, IL-3, IL5, and GM-CSF in the hippocampus.

Each data point represents the mean of 8–10 mice; error bars are the SEM; P < 0.05.

Figure 7:. Frequency of licking during 90-min recording sessions that were preceded by 24 h of food and water deprivation.

(A) Eight and 10 traces are shown from ThioTEPA and control mice, respectively. The frequency histograms were binned at 1 s. ThioTEPA mice showed a non-significant increase in licking at the beginning of the recording session. (B) Averaged frequency histograms from each group of mice. Top panel: Group data (mean + SEM) frequencies were calculated after binning at 1 s and smoothing with 181 s of adjacent averaging. Lower panel: Group data (mean ± SEM) were calculated using long-duration bins (1 min) and adjacent averaging of 3 min. Each graph represents the mean of 8–10 mice.

Figure 8:. ILI distribution histograms.

(A) The histograms were generated for ILIs between 60 ms and 1000 ms and binned at 1 ms (only the range of 60–300 ms is shown). Then, they were smoothed by adjacent averaging of 21 ms (i.e. each bin is the average of itself, 10 bins before it, and 10 bins after) and normalized to the total number of ILIs (between 60 ms and 1000 ms). Each panel represents histograms obtained from one group of mice, as indicated. (B) Superimposed average histograms of ILIs for comparing the 2 groups. (C) Representative licking traces from a ThioTEPA-treated mouse and saline mouse. Note that the ThioTEPA-treated mouse has more irregular ILIs with missing licks. Each graph represents the mean of 8–10 mice.

Figure 9:. Differences in the ILI distribution histograms between mice treated with ThioTEPA or saline.

Data (means ± SEM) were collected during 90-min recording sessions and averaged from 8 mice (ThioTEPA group) and 10 mice (control group). P values indicate significant differences between treated and control groups (independent t-test). (a) Mean licking frequency over the entire recording session indicates that TH-treated mice and mice did not a significant difference in the number of licks produced during the 90-min session. (b) A schematic representation of the statistical parameters that were quantified in panels (c-i) (see Materials and methods for additional details). (c-i) Group data of the different statistical parameters that characterize the ILI distribution histograms. Each graph represents the mean of 8–10 mice.