Caffeine and Modafinil Ameliorate the Neuroinflammation and Anxious Behavior in Rats during Sleep Deprivation by Inhibiting the Microglia Activation

Abstract

Background: Sleep deprivation (SD) plagues modern society due to the professional demands. It prevails in patients with mood and neuroinflammatory disorders. Although growing evidence suggests the improvement in the cognitive performance by psychostimulants during sleep-deprived conditions, the impending involved mechanism is rarely studied. Thus, we hypothesized that mood and inflammatory changes might be due to the glial cells activation induced modulation of the inflammatory cytokines during SD, which could be improved by administering psychostimulants. The present study evaluated the role of caffeine/modafinil on SD-induced behavioral and inflammatory consequences. Methods: Adult male Sprague-Dawley rats were sleep deprived for 48 h using automated SD apparatus. Caffeine (60 mg/kg/day) or modafinil (100 mg/kg/day) were administered orally to rats once every day during SD. Rats were subjected to anxious and depressive behavioral evaluation after SD. Subsequently, blood and brain were collected for biochemical, immunohistochemical and molecular studies. Results: Sleep deprived rats presented an increased number of entries and time spent in closed arms in elevated plus maze test and decreased total distance traveled in the open field (OF) test. Caffeine/modafinil treatment significantly improved these anxious consequences. However, we did not observe substantial changes in immobility and anhedonia in sleep-deprived rats. Caffeine/modafinil significantly down-regulated the pro- and up-regulated the anti-inflammatory cytokine mRNA and protein expression in the hippocampus during SD. Similar outcomes were observed in blood plasma cytokine levels. Caffeine/modafinil treatment significantly decreased the microglial immunoreactivity in DG, CA1 and CA3 regions of the hippocampus during SD, however, no significant increase in immunoreactivity of astrocytes was observed. Sholl analysis signified the improvement in the morphological alterations of astrocytes and microglia after caffeine/modafinil administration during SD. Stereological analysis demonstrated a significant improvement in the number of ionized calcium binding adapter molecule I (Iba-1) positive cells (different states) in different regions of the hippocampus after caffeine or modafinil treatment during SD without showing any significant change in total microglial cell number. Eventually, the correlation analysis displayed a positive relationship between anxiety, pro-inflammatory cytokines and activated microglial cell count during SD. Conclusion: The present study suggests the role of caffeine or modafinil in the amelioration of SD-induced inflammatory response and anxious behavior in rats. Highlights - SD induced mood alterations in rats. - Glial cells activated in association with the changes in the inflammatory cytokines. - Caffeine or modafinil improved the mood and restored inflammatory changes during SD. - SD-induced anxious behavior correlated with the inflammatory consequences.

Keywords: caffeine; cytokines; microglia; modafinil; mood changes; neuroinflammation; sleep deprivation.

Figure 1

Schematic design of the experiment.

Figure 2

Assessment of the anxious behavior following caffeine/modafinil treatment during sleep deprivation (SD). (A) Track plot of the rats during elevated plus maze (EPM) test and the study parameters: (B) proportion of the number of entries in the open arms; (C) proportion of the time spent in the open arms. (D) Track plot of the rats during open field (OF) test; (E) total distance traveled in the OF. *p < 0.05 when compared to control treated with vehicle; ^φp < 0.05 when compared to sleep deprived treated with vehicle. Two way ANOVA followed by Tukey post hoc test with multiple comparison was used.

Figure 3

Assessment of the depressive behavior following caffeine/modafinil treatment during SD. (A) Track plot of the rats during forced swim test (FST) test; (B) total immobility time; (C) sucrose preference over the total volume of fluid intake. Two way ANOVA followed by Tukey post hoc with multiple comparison test was applied for statistical comparison between groups. Blue dot represented the starting point and the red dot represented the end point of the test.

Figure 4

Fold changes in the inflammatory cytokines in hippocampus during caffeine/modafinil administration following SD. The concentration of cytokine levels were measured in picograms per milliliter and expressed as the fold changes in TNF-α (A,F); interleukin-1β (IL-1β) (B,G); IL-6 (C,H; pro-inflammatory cytokines), IL-4 (D,I); IL-10 (E,J; anti-inflammatory cytokines) in the hippocampus. *p < 0.05 when compared to control treated with vehicle; ^φp < 0.05 when compared to sleep deprived treated with vehicle. Two way ANOVA followed by Tukey post hoc test with multiple comparison were used for the statistical evaluation.

Figure 5

Caffeine or modafinil treatment inhibited the glial cell immunoreactivity in rat hippocampus following SD. (A) Representable image of astrocytes expression in DG, CA1 and CA3 regions of the hippocampus. Glial fibrillary acidic protein (GFAP) immunoreactivity quantification in (B) DG region; (C) CA1 region; (D) CA3 region of the hippocampus. (E) Representable image of microglial cells expression in DG, CA1 and CA3 regions of the hippocampus. Ionized calcium binding adapter molecule I (Iba-1) cell immunoreactivity quantification in (F) DG region; (G) CA1 region; (H) CA3 region of the hippocampus. *p < 0.05 when compared to control treated with vehicle; ^φp < 0.05 when compared to sleep deprived treated with vehicle. Two way ANOVA followed by Tukey post hoc multiple comparison test was applied for statistical comparison between groups and for the graphical representation, values expressed mean percentage of Control ± SEM.

Figure 6

Caffeine or modafinil administration altered the morphology of astrocytes and microglia towards the resting state in rat hippocampus during SD. Representable image (intersection and segmented mask) of (A) normal and activated astrocyte; (B) resting, intermediate and activated stage microglial cell. Changes in the (C) soma density; (D) soma area; (E) sum inters; (F) mean inters; (G) ramification index; (H) astrocyte length of astrocytes in DG, CA1 and CA3 regions of the hippocampus. Changes in the (I) soma density; (J) soma area; (K) sum inters; (L) mean inters; (M) ramification index; (N) microglial length of microglia cells. *p < 0.05 when compared to control treated with vehicle; ^φp < 0.05 when compared to sleep deprived treated with vehicle. Two way ANOVA followed by Tukey post hoc test with multiple comparison was applied for statistical comparison between groups and for the graphical representation, values expressed mean percentage of Control ± SEM.

Figure 7

Caffeine or modafinil improved the microglial cell numbers following SD. Changes in the microglial cell count at different stages (resting, intermediate and activated) in (A) DG region; (B) CA1 region; (C) CA3 region of the hippocampus. Total microglial cell count in (D) DG region; (E) CA1 region; (F) CA3 region of the hippocampus. *p < 0.05 when compared to control treated with vehicle; ^φp < 0.05 when compared to sleep deprived treated with vehicle. Two way ANOVA followed by Tukey post hoc test with multiple comparison was applied for statistical comparison between groups and for the graphical representation, values expressed mean percentage of Control ± SEM.

Figure 8

Correlation analysis predicting the interaction between the anxious behavior, pro-inflammatory cytokines and activated microglia cell during SD. Interaction between the anxious behavior and activated microglial cell as shown by correlation between (A) proportion of the number of entries in the open arms and activated microglial cell count in the hippocampus; (B) proportion of the time spent in the open arms and activated microglial cell count in the hippocampus; (C) total distance traveled in the OF and activated microglial cell count in the hippocampus. Finally, the interaction between the anxious behavior and pro-inflammatory cytokine levels as shown by correlation between (D) proportion of the number of entries in the open arms and TNF-α level in hippocampus; (E) proportion of the time spent in the open arms and TNF-α level in hippocampus; (F) total distance traveled in the OF and TNF-α level in hippocampus; (G) proportion of the number of entries in the open arms and IL-1β level in hippocampus; (H) proportion of the time spent in the open arms and IL-1β level in hippocampus; (I) total distance traveled in the OF and IL-1β level in hippocampus. Pearson’s test was applied for correlation analysis. p < 0.05 was considered to be statistically significant.