Severity of enteric nervous system neuropathy during Herpes simplex virus type 1 infection is related to viral replication

Background. Recently, we have shown that HSV1 establishes a latent infection in the mouse enteric nervous system (ENS), leading to gut dysmotility with no signs of illness.(1) Aim. Considering that ICP27, a HSV1 immediate early gene, is involved in viral DNA replication (2), this study aimed to assess the effects of HSV-1 replication on ENS morphology and intestinal contractility in mice infected with wild type (WT) and ICP27-null (KO) HSV1. Methods. In C57/Bl6 mice (males, body weight=24\ub12 g) infection with WT or KO HSV1 was established by intranasal viral dose (103 pfu) followed 4 weeks (W) later by an intragastric inoculum (IG; 108 pfu).1 Mice (n=6-8) were sacrificed 1, 4 and 8W after IG and the presence of WT and KO HSV1 infection was shown by PCR amplification of HSV1-tk gene in brain and myenteric ganglia (MG). In isolated ileal segments changes in tension were recorded isometrically following electric field stimulation (EFS, 1-40 Hz). Alterations of ENS morphology of whole mount preparations of ileal longitudinal muscle-myenteric plexus (LMMPs), immunostained with HuC/D, peripherin, or nNOS (neuronal Nitric Oxide Synthetase) was assessed by confocal microscopy. For each experimental point 5 LMMPs were examined and 5 microscopic fields were captured to evaluate immunostained cells numbers. Cholinergic network was investigated by analyzing LMMPs, biochemically stained with acetylcholinesterase (AChE) and evaluated in 8 random fields by optical microscopy. Data are reported as percentage of mean \ub1 standard error and statistically analyzed by unpaired Student\u2019s t test. P<0.05 were considered statistically significant. Results. In the brain and ENS WT and KO HSV1 established a latent infection shown by the presence of viral tk-DNA. In the ENS of WT HSV1 infected mice analyses of LMMPs double-immunostained with HuC/D and nNOS revealed an altered number of HuC/D+ and nNOS+ cells at 1W (-37\ub112% and +60\ub120%, respectively; P<0.05 vs sham) and 8W (-32\ub110% and +99\ub121%, respectively; P<0.05 vs sham) after IG infection whereas KO HSV1 infection reduced the number of HuC/D+ cells at 1W PI (-43\ub110%) with no changes in the number of nNOS+ neural cells. By performing peripherin labelling an irregular distribution and interrupted neuronal processes was found in MG after 8W of WT HSV1 infection. AChE staining revealed a significant reduction in the density of cholinergic processes after 1W of WT HSV1 infection. In KO HSV1 infected mice no changes in peripherin immunoreactivity were found at 1-8 W PI whereas AChE distribution showed a reduction at 1W and a significant increase at 4 and 8W PI in cholinergic fibers density. Neurally-mediated contractions induced by EFS (20 Hz) were significantly reduced at 1W (-52\ub117%; P<0.05 vs sham) and increased at 8W (+43\ub120%; P