Altered Brain Excitability and Increased Anxiety in Mice With Experimental Colitis: Consideration of Hyperalgesia and Sex Differences

Abstract

Crohn's disease (CD) and ulcerative colitis (UC) are incurable lifelong inflammatory bowel diseases (IBD) with a rising worldwide incidence. IBD is characterized by diarrhea, rectal bleeding, severe cramping and weight loss. However, there is a growing evidence that IBD is also associated with anxiety- and depression-related disorders, which further increase the societal burden of these diseases. Given the limited knowledge of central nervous system (CNS) changes in IBD, we investigated CNS-related comorbidities in a mouse model of experimental colitis induced by dextran sulfate sodium (DSS) administration in drinking water for 5 days. In male and female C57BL6J mice, DSS treatment caused increased brain excitability, revealed by a decrease in seizure onset times after intraperitoneal administration of kainic acid. Moreover, both sexes showed increased anxiety-related behavior in the elevated plus-maze (EPM) and open field (OF) paradigms. We assessed somatic pain levels, because they may influence behavioral responses. Only male mice were hyperalgesic when tested with calibrated von Frey hairs and on the hotplate for mechanical and thermal pain sensitivity respectively. Administration of diazepam (DZP; ip, 1 mg/kg) 30 min before EPM rescued the anxious phenotype and improved locomotion, even though it significantly increased thermal sensitivity in both sexes. This indicates that the altered behavioral response is unlikely attributable to an interference with movement due to somatic pain in females. We show that experimental colitis increases CNS excitability in response to administration of kainic acid, and increases anxiety-related behavior as revealed using the EPM and OF tests.

Keywords: IBD; anxiety; colitis; diazepam; pain; sex differences.

Figure 1

Increased brain excitability in dextran sulfate sodium (DSS) colitis. Five days 2.5% DSS treatment decreased the seizure onset time in both males (A) and females (C) on day 7 (D7), and did not correlate with the macroscopic damage scores in males (B) nor females (D). ***P < 0.001 vs. control mice.

Figure 2

DSS colitis increases anxiety-related behavior and reduces locomotion on the elevated plus-maze (EPM). Male and female control/DSS-treated mice were tested on the EPM on D7. DSS-induced colitis resulted in an anxiety-like phenotype as indicated by reduced percentage time in the unprotected open (A,D) and increased time in the closed arm (B,E) respectively. Reduced locomotor activity is indicated as the reduced total distance traveled in the maze (C,F) in both DSS-treated males and females. ***P < 0.001, **P < 0.01, *P < 0.05 vs. control mice.

Figure 3

DSS colitis increases anxiety-related behavior and reduces locomotion in the open field (OF). Male and female control/DSS-treated mice, previously tested on the EPM (D7) were subjected to the OF test the next day (D8). In line with previous D7 observations on the EPM, the DSS-induced anxiety-like phenotype remained as indicated by the increased percent time in the peripheral zone of the box (A,D), and reduced center zone percent time (B,E) in both sexes. The reduced total distance traveled (C,F) was used as a measure of locomotor activity in both males and females. ***P < 0.001, **P < 0.01, vs. control mice.

Figure 4

Diazepam (DZP) rescues the anxious phenotype and locomotion in DSS-treated animals. DZP treatment, given 30 min before the EPM test to DSS-treated animals reduced anxiety-related behavior, reflected by the increased percent time spent in the open (A,D) and reduced closed arm percent time (B,E) in both males (A,B) and females (D,E). DZP treatment increased locomotor activity in DSS-treated males (C) and rescued (F) the decreased locomotion previously seen in DSS-treated females (Figures 2F, 3F). **P < 0.01, *P < 0.05 vs. DSS + VEH mice.

Figure 5

Mechanical and thermal hyperalgesia in DSS colitis: aggravation by DZP. On the morning of D7, separate cohorts of control and DSS-treated mice were tested for mechanical and thermal pain perception by subjecting their paws to calibrated von Frey hairs, or exposing them to the hot plate (52°C) for a maximum of 30 s respectively. DSS treatment reduced withdrawal thresholds (A), and the foot withdrawal latency (B) in only males and not females (D,E). A different set of DSS-treated mice were administered VEH or DZP (ip) 30 min before testing. DZP aggravated hyperalgesia by further reducing the foot withdrawal latency in both males (C) and females (F). ***P < 0.001, *P < 0.05 vs. control/DSS vehicle mice.