Department of Veterinary Pathology1 and Department of Preclinical Veterinary Sciences2, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK
Author for correspondence: Robert Dalziel.Fax +44 131 650 6511. e-mail R.G.Dalziel{at}ed.ac.uk
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Abstract |
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In latent herpes simplex virus type 1 (HSV-1) infections no viral proteins are detected in the latently infected cells and the only viral transcripts detected are the latency-associated transcripts (LATs) (Stevens et al., 1987 ). These LATs are stable intron sequences whose function is unclear and from which no protein is known to be translated. In contrast, five VZV-encoded transcripts and proteins (genes 4, 21, 29, 62 and 63) have been shown to be present in human and rat ganglia with latent VZV infection (Lungu et al., 1998
; Sadzot-Delvaux et al., 1990
; Cohrs et al., 1996
). The role, if any, of these proteins in PHN is unknown.
We utilized a previously reported animal model of latent VZV infection (Sadzot-Delvaux et al., 1990 ) to investigate if rats infected with VZV exhibit alterations in sensory responses similar to those observed in PHN. VZV (Dumas et al., 1981
) was propagated on CV-1 cells and harvested when cells exhibited approximately 80% cytopathic effect. Male Wistar rats (weight 220450 g, n=11) were anaesthetized with Sagatal [60 µl/100 g, intraperitoneal (i.p.)] and injected subcutaneously in the left (ipsilateral) glabrous footpad with approximately 4x106 infected cells per animal in 50 µl PBS, as previously described (Sadzot-Delvaux et al., 1995
). Control rats (n=10) were injected with mock-infected CV-1 cells and housed separately from virus-infected animals. Behavioural tests were carried out 110 days prior to infection and then up to 33 days post-infection to test for the development of hyperalgesia and allodynia, and consisted of the following.
1. Paw withdrawal in response to graded innocuous and noxious mechanical stimuli. A calibrated set of von Frey nylon monofilaments, each of which exerts a known force at its bending threshold (1·5125·9 g), was used to determine the mean mechanical force (g/unit area) to cause two or more reflex foot withdrawals over ten applications of graded filaments. The stimulus was applied at 12 s intervals to the ventral, glabrous surface of both the left and right hind paws (Chaplan et al., 1994 ) and repeated three times with a 5 min interval between tests.
2. Paw withdrawal to noxious thermal stimuli (Hargreaves et al., 1998 ). The mean latency (to the nearest 0·1 s) for hind paw withdrawal to noxious heat (range 3055 °C) applied to the glabrous skin of the footpad was assessed using a Ugo Basile Unit. A standard cut-off latency of 15 s prevented possible tissue damage.
Statistical analysis was carried out using the Wilcoxon test for left to right hind limb comparisons, within groups, or the MannWhitney U-test for between group comparisons. Care was taken to ensure that there was no undue bias in the way the testing procedure was performed by not allowing the animal to put its weight on the hind paw being tested (Kauppila et al., 1998 ). At the end of 33 days, animals were killed and tissues taken for immunohistochemical processing.
Animals showed no obvious signs of discomfort and phenotypic changes were only evident on formal testing. For VZV-injected rats the thresholds for both the von Frey filament (Fig. 1B) and noxious thermal tests (Fig. 1D
) showed a significant and sustained decline for the injected left hind paw compared to the uninjected right hind paw (P<0·05; Wilcoxon test, n=5) for up to 33 days following injection. This alteration in sensory phenotype was observed in all infected animals as indicated by the error bars in Fig. 1
. In contrast, control mock-infected rats showed no significant difference between the injected and uninjected hind paw responses over this time period, or compared to preinjection control tests, (Fig. 1A
, C
; Wilcoxon test, n=5).
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To monitor VZV protein expression in latently infected nervous tissue immunohistochemical analysis of the lumbar dorsal root ganglia was carried out. Animals were deeply anaesthetized with Sagatal (120 µl/100 g, i.p.) and perfused transcardially with 0·1 M phosphate-buffered saline (PBS, pH 7·4; containing 3 mM sodium nitrite and 1000 U heparin) before being perfused with 4% paraformaldehydePBS. Dorsal root ganglia taken from lumbar segments L3L6 were removed and post-fixed in the same solution overnight. Tissue was then transferred to a 25% sucrose solution for a further 24 h, prior to being stored in cryoprotectant (30% ethylene glycol, 20% glycerol, in PBS, pH 5·5) at 4 °C until ready for sectioning. DRG from several infected animals were pooled, embedded in paraffin wax and 5 µM sections cut and mounted onto Vectabond-coated slides. Uninfected animals were treated in a similar manner. Sections were dewaxed and rehydrated, followed by a brief rinse in PBS prior to incubation with rabbit anti-IE 63 [a kind gift of C. Sadzot-Delvaux (Sadzot-Delvaux et al., 1995 )]. Bound antibody was detected using the Vectastain Elite ABC kit and immunoreactivity visualized by the 3,3'-diaminobenzidine tetrachlorideimmunoperoxidase method. Finally, sections were lightly counterstained with 1% eosin.
The presence of IE 63 protein was demonstrated in ipsilateral lumbar dorsal root ganglia in VZV-injected rats compared to control, uninfected rats (Fig. 2). No evidence of inflammatory infiltrates or of neuronal cell death was observed in the DRG sections. Thus, in this model, it would appear that the presence of VZV in DRG correlates with an increased sensitivity to sensory stimuli.
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References |
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Received 25 February 1999;
accepted 3 June 1999.