Autonomic nervous dysfunction in hamsters infected with West Nile virus

Abstract

Clinical studies and case reports clearly document that West Nile virus (WNV) can cause respiratory and gastrointestinal (GI) complications. Other functions controlled by the autonomic nervous system may also be directly affected by WNV, such as bladder and cardiac functions. To investigate how WNV can cause autonomic dysfunctions, we focused on the cardiac and GI dysfunctions of rodents infected with WNV. Infected hamsters had distension of the stomach and intestines at day 9 after viral challenge. GI motility was detected by a dye retention assay; phenol red dye was retained more in the stomachs of infected hamsters as compared to sham-infected hamsters. The amplitudes of electromygraphs (EMGs) of intestinal muscles were significantly reduced. Myenteric neurons that innervate the intestines, in addition to neurons in the brain stem, were identified to be infected with WNV. These data suggest that infected neurons controlling autonomic function were the cause of GI dysfunction in WNV-infected hamsters. Using radiotelemetry to record electrocardiograms and to measure heart rate variability (HRV), a well-accepted readout for autonomic function, we determined that HRV and autonomic function were suppressed in WNV-infected hamsters. Cardiac histopathology was observed at day 9 only in the right atrium, which was coincident with WNV staining. A subset of WNV infected cells was identified among cells with hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4) as a marker for cells in the sinoatrial (SA) and atrioventricular (AV) nodes. The unique contribution of this study is the discovery that WNV infection of hamsters can lead to autonomic dysfunction as determined by reduced HRV and reduced EMG amplitudes of the GI tract. These data may model autonomic dysfunction of the human West Nile neurological disease.

Figure 1. Gastrointestinal motility in WNV-infected hamsters.

A) Gastrointestinal distension in WNV- or sham-infected hamsters 9 days after s.c. viral challenge (arrows). B) WNV- or sham-infected hamsters fasted for 15 hr were administered 1.5 mL of phenol red solution at 7–9 days after s.c. viral challenge. Upon necropsy, relative amounts of phenol red dye were measured in the stomachs. The higher levels of phenol red dye indicated a reduced gastrointestinal motility. (**P≤0.01 using a two-way t test.)

Figure 2. Electromyography (EMG) of intestines in WNV-infected hamsters.

Hamsters were surgically implanted with telemetry transmitters with electrodes attached to the duodenum just below the pyloric sphincter, and then injected s.c with WNV or sham. At 9 days after viral challenge, EMGs were measured continuously for 4 min in isoflurane-anesthetized hamsters that had fasted for 15 hr. At 2 min (arrows), hamsters were orally gavaged with 1.5 mL of 5% glucose. Representative EMG tracings of A) sham-injected and B) WNV-injected hamsters. C) Mean amplitudes ± SD from 0–2 min. (fasting) and 2–4 min. (***P≤0.001 using a two-way t test.)

Figure 3. WNV-immunostaining and histopathological immunostaining of the GI tract in WNV-infected hamsters.

Hamsters were injected s.c. with WNV or sham, and necropsied 9 days later. The intestine (duodenum, jejunum, ileum, colon) was sectioned for A) fluorescent immunohistochemical co-staining of WNV envelope and NSE and DAPI (nuclear), and B) WNV envelope and synaptophysin and DAPI. C) H&E staining of a representative section of intestine is shown. WNV-stained cells (white arrow). histopathological lesion (black arrow). Bar scale 100 µm.

Figure 4. Nerve conduction velocity (NCV) in WNV- or sham-infected hamsters.

Hamsters were injected s.c with WNV or sham (cell culture supernatant), and the NCV was recorded at days 0, 7, and 10 from the A) sciatic-tibial nerve, B) median sensory nerve. C) As a positive control to see if the procedure could detect differences in nerve conduction velocity, NCV was measured in uninfected or sham-infected hamsters injected i.m. with 2% lidocaine or saline in the vicinity sciatic-tibial nerve one day earlier. (*P≤0.05 using one-way analysis of variance.)

Figure 5. Heart rate variability (HRV) in alert WNV-infected hamsters.

Hamsters were surgically implanted with telemetry transmitters with electrodes attached to both pectoralis major muscles, and then injected s.c with WNV (n = 6) or sham (n = 6). ECGs were measured continuously over the course of the experiment. Data were extracted at 0, 4, 7, and 9 days after viral challenge to calculate changes in HRV evaluated in both the A) time domain and B) frequency domain. A) Time domain calculations: R-R intervals (RR); standard deviation of normal RR (SDNN), root mean square of the differences between consecutive RR (RMSSD); number of successive RR interval pairs that differ more than 50 ms (NN50), and NN50 divided by the total number of RR intervals (pNN50) ± SD. B) Frequency domain calculations: Mean low frequency (LF) % total power, high frequency (HF) % total power; and LF/HF ratio of the absolute values ± SD. (*P≤0.05, **P≤0.01, ***P≤0.001 using two-way analysis of variance with the Bonferroni post-test to compared groups at each day or t tests comparing WNV- and sham-data on the same day.)

Figure 6. WNV-immunostaining and histopathological immunostaining of the heart in WNV-infected hamsters.

Hamsters were injected s.c. with WNV or sham, and necropsied 9 days later. The heart (right atrium, left atrium, right ventricle, and left ventricle) was sectioned for A) H&E staining of the vena cava of the right atrium (A, upper panel), and WNV envelope staining of the vena cava (A, lower panel). Histopathology was only observed in the right atrium, not in other portions of the heart, e.g. intraventricular septum (A, right in lower panel). Histopathological lesions (arrows). B) Immunofluorescent staining of WNV envelope and HCN4 of SA and AV nodes in the right atrium. Bar scale 100 µm.