Tick-borne encephalitis affects sleep-wake behavior and locomotion in infant rats

Abstract

Background/aims: Tick-borne encephalitis (TBE) is a disease affecting the central nervous system. Over the last decade, the incidence of TBE has steadily increased in Europe and Asia despite the availably of effective vaccines. Up to 50% of patients after TBE suffer from post-encephalitic syndrome that may develop into long-lasting morbidity. Altered sleep-wake functions have been reported by patients after TBE. The mechanisms causing these disorders in TBE are largely unknown to date. As a first step toward a better understanding of the pathology of TBEV-inducing sleep dysfunctions, we assessed parameters of sleep structure in an established infant rat model of TBE.

Methods: 13-day old Wistar rats were infected with 1 × 10⁶ FFU Langat virus (LGTV). On day 4, 9, and 21 post infection, Rotarod (balance and motor coordination) and open field tests (general locomotor activity) were performed and brains from representative animals were collected in each subgroup. On day 28 the animals were implanted with a telemetric EEG/EMG system. Sleep recording was continuously performed for 24 consecutive hours starting at day 38 post infection and visually scored for Wake, NREM, and REM in 4 s epochs.

Results: As a novelty of this study, infected animals showed a significant larger percentage of time spend awake during the dark phase and less NREM and REM compared to the control animals (p < 0.01 for all comparisons). Furthermore, it was seen, that during the dark phase the wake bout length in infected animals was prolonged (p = 0.043) and the fragmentation index decreased (p = 0.0085) in comparison to the control animals. LGTV-infected animals additionally showed a reduced rotarod performance ability at day 4 (p = 0.0011) and day 9 (p = 0.0055) and day 21 (p = 0.0037). A lower locomotor activity was also seen at day 4 (p = 0.0196) and day 9 (p = 0.0473).

Conclusion: Our data show that experimental TBE in infant rats affects sleep-wake behavior, leads to decreased spontaneous locomotor activity, and impaired moto-coordinative function.

Keywords: Anxiety-like behavior; Chemokines and cytokines; Infant rats; Langat virus; Locomotion; Neurofilament; Sleep; Sleep–wake behavior; Tick-borne encephalitis.

Fig. 1

Experimental setting for Telemetry and EEG/EMG recording and exemplary labeled EEG/EMG data: A the placement of the telemetry body in the abdomen. B Representation of an animal head with implanted EEG and EMG electrodes. C Electrode (EEG and EMG) placement in the skull and neck muscle of the animal (not to scale). D Schematic displaying EEG/EMG recording. Two stacked SmartPads are used to allow Cohousing in a cage. The primary SmartPad is needed for wireless power to the implanted telemeters whereas each implanted sensor transmits the data separately through the two pads. E Exemplary data of a Wake stretch, which is characterized by high and variable EMG activity and medium EEG activity. F Exemplary data of a NREM sleep phase, characterized by low EMG activity and prominent EEG activity in the delta range. G Example of a REM sleep phase characterized very low EMG activity and medium EEG activity mainly in the theta band

Fig. 2

Viral load in brain tissue and increased CSF chemokines, cytokines and NFL in infected animals: A Virus titer at day 4 (n_cerebellum = 11; n_{midbrain/forebrain} = 12), day 9 (n_cerebellum = 13; n_{midbrain/forebrain} = 14), day 21 (n = 9) and after the sleep measurement (n = 6). B–F Chemokines and Cytokines assessed in the CSF of control and infected. G Nfl level assessed in CSF in control and infected animals, with significantly higher level in the infected animals at Day 9. †p < 0.1, *p < 0.05, **p < 0.01

Fig. 3

Reduced performance in the behavioral tests assessing motor coordination and locomotion following LGTV infection: A Rotarod test showing the latency to fall in seconds (day 4: n_control = 31, n_infection = 36; day 9: n_control = 22, n_infection = 26; day 21: n_control = 15, n_infection = 14), B and C Open field tests (day 4: n = 24; day 9: n = 20; day 21: n_control = 13, n_infection = 14) showing the average distance walked in cm and the percentage of the time spent in the center of the arena, respectively. †p < 0.1, *p < 0.05, **p < 0.01

Fig. 4

Extensive changes to the sleep structure determination by EEG/EMG after the LGTV infection: Sleep macrostructure of control (A) and infected (B) animals over 24 h. C Overview of the percentage in wake, NREM, and REM phases during light and dark period in control and infected animals. D–F Percentage of epochs displayed in 3 h bins for the wake, NREM, and REM phases, confirming that the infected animals spent more time awake than the control animals. G Mean number of state changes. H Mean duration of wake stage. I Correlation between sleep markers and the rotarod performance at Day 21. J Delta power during NREM in the light and dark period. K Theta power during REM for the light and dark period. n_control = 8, n_infection = 10; †p < 0.1, *p < 0.05, **p < 0.01