Clock gene expression during chronic inflammation induced by infection with Trypanosoma brucei brucei in rats

Abstract

African sleeping sickness is characterized by alterations in rhythmic functions. It is not known if the disease affects the expression of clock genes, which are the molecular basis for rhythm generation. We used a chronic rat model of experimental sleeping sickness, caused by the extracellular parasite Trypanosoma brucei brucei (Tb brucei), to study the effects on clock gene expression. In tissue explants of pituitary glands from Period1-luciferase (Per1-luc) transgenic rats infected with Tb brucei, the period of Per1-luc expression was significantly shorter. In explants containing the suprachiasmatic nuclei (SCN), the Per1-luc rhythms were flat in 21% of the tissues. We also examined the relative expression of Per1, Clock, and Bmal1 mRNA in the SCN, pineal gland, and spleen from control and infected rats using qPCR. Both Clock and Bmal1 mRNA expression was reduced in the pineal gland and spleen following Tb brucei infection. Infected rats were periodic both in core body temperature and in locomotor activity; however, early after infection, we observed a significant decline in the amplitude of the locomotor activity rhythm. In addition, both activity and body temperature rhythms exhibited decreased regularity and "robustness." In conclusion, although experimental trypanosome infection has previously been shown to cause functional disturbances in SCN neurons, only 21% of the SCN explants had disturbed Per1-luc rhythms. However, our data show that the infection overall alters molecular clock function in peripheral clocks including the pituitary gland, pineal gland, and spleen.

Figure 1

Effects of Tb brucei infection on the circadian rhythms of Per1-luc expression. (A) Representative Per1-luc expression rhythms in isolated SCN tissue explants recovered from control (black trace) or Tb brucei–infected (gray trace) rats in 12:12-hour LD. (B) In 3 of the SCN explants, rhythms were blunted and/or absent. (C) Representative Per1-luc expression rhythms in isolated pituitary tissue explants recovered from control (black trace) or Tb brucei (gray trace). The period (insert) of Per1-luc expression in pituitary glands from animals maintained in LD was significantly shorter in Tb brucei infection, indicated by the asterisk. The gray-black bars at the bottom of the figures indicate the LD cycle prior to sacrifice.

Figure 2

Relative amounts of (A) Clock in the pineal gland and (B) Bmal1 in spleen sampled from control and infected rats at ZT 6 to 7 (Day cont; tryp) and ZT 18 to 19 (Night cont; tryp). Two-way ANOVA (2wA) showed significant differences between control and infected groups.

Figure 3

Example of actograms obtained from wheel-running activity in a 12:12-hour light:dark cycle from 1 control rat and 2 rats infected with Tb brucei, respectively. The record from the first infected rat (“Tb brucei 1”) is representative for the infected group; however, the second record shown (“Tb brucei 2”) demonstrates arrhythmicity in one infected rat at the end of the activity recording period. The top horizontal bar indicates the time of subjective night (black) and day (gray).

Figure 4

Example of double-plotted actograms obtained from wheel-running activity in constant darkness from (A) 1 control and (B) 1 infected rat, respectively. The rats were first held in a 12:12-hour LD cycle, indicated by the bars, before released into constant darkness (arrows).

Figure 5

Core body temperature records from 1 control (A-C) and 1 infected (D-F) rat. B and E show magnifications of postinfection days 20 to 30, and C and F show magnifications of postinfection days 40 to 50.