ABSTRACT
Title
Herpes Simplex virus type-1 infection of the enteric nervous system leads to gut neuroanatomical and neurochemical abnormalities
Authors
M. Qesari
Doctorate School in Pharmacology, Toxicology and Therapy
Dept. of Pharmacology and Anaesthesiology - University of Padova, Italy
Doctorate School in Pharmacology, Toxicology and Therapy
Dept. of Pharmacology and Anaesthesiology - University of Padova, Italy
Abstract
The enteric nervous system (ENS) plays a pivotal role in the regulation of intestinal motility. Neurotropic viruses have been proposed as possible etiological factors able to compromise the integrity of ENS either directly or through immuno-mediated mechanisms, leading to gastrointestinal motor disorders (GIMDs) [1]. Recently, we have shown that Herpes Simplex virus type-1 (HSV-1), orally inoculated to rodents, infects the ENS and affects gut motor function without signs of systemic infection [2]. The aim of the present study was to identify neuroanatomical and neurochemical alterations in the ENS at 1, 4 and 8 weeks (W) after HSV-1 infection.
Male mice C57Bl/6 (body weight=20±2 g; Harlan SpA, Italy) were inoculated with 103 p.f.u HSV-1 intranasally (IN) and after 4 W with 108 p.f.u. HSV-1 intragastrically (IG). After 1-8 W, HSV-1 infection was determined in the brain and in freshly isolated myenteric ganglia by molecular analysis. The neurochemical phenotype of the ENS was analyzed with antibodies against Hu C/D (somata neuronal marker), S100 β (glial marker), peripherin (process and somata neuronal marker), nNOS (nitrergic neuronal marker) and with acetylcholine esterase (AChE) staining in ileum whole mount preparations. Morphoquantitative analysis of myenteric ganglia were performed in 5 random ganglionic fields for each mouse using confocal microscopy and Nis Elements vs. 3.0 as image analysis system. The cholinergic network of longitudinal muscle-myenteric plexus was evaluated in 8 random fields by optical microscopy and the following optical density distribution of AChE by ImageJ vs. 1.14 program. Protein expression of peripherin, nNOS and pAKT was assessed by western blot analysis.
A significant reduction of the ganglionic areas in the myenteric plexus was found in mice 1 (-33%) and 8 W (-18%) postinfection (PI). S-100 β positive glial cells increased from 22±3 insham to 36±4 inmice 4 W PI. In the ENS the Hu C/D labelling was significantly decreased at 1 W PI (-50%) and further reduced at 8 W after HSV-1 infection. By performing peripherin staining an irregular distribution and interrupted neurofilaments were found at 1 and 8 W PI whereas protein levels were significantly increased (+42%) at 1 W PI. In longitudinal muscle-myenteric plexus preparations, nNOS protein levels were significantly increased at 1 and 4 W (+56% and +37%, respectively) post IG inoculum, confirming an altered NANC inhibitory pathway. Akt phosphorylation was also significantly increased during viral infection. Furthermore, a reduced staining of acetylcholine esterase-positive neurons, large fibers and small fibers, was evident at 1 and 8 W PI.
These results provide evidence that HSV-1 induced neuronanatomical and neurochemical alterations in the ENS, leading to a chronic neuropathy. HSV-1 infection of ENS appears to be a suitable animal model for studying the role of ENS in the onset of GIMDs.
1. Gesser RM, Koo SC (1996) J Viriol 70: 4097-102
2. Brun P et al. (2010) Gastroenterology 138:1790-801
Male mice C57Bl/6 (body weight=20±2 g; Harlan SpA, Italy) were inoculated with 103 p.f.u HSV-1 intranasally (IN) and after 4 W with 108 p.f.u. HSV-1 intragastrically (IG). After 1-8 W, HSV-1 infection was determined in the brain and in freshly isolated myenteric ganglia by molecular analysis. The neurochemical phenotype of the ENS was analyzed with antibodies against Hu C/D (somata neuronal marker), S100 β (glial marker), peripherin (process and somata neuronal marker), nNOS (nitrergic neuronal marker) and with acetylcholine esterase (AChE) staining in ileum whole mount preparations. Morphoquantitative analysis of myenteric ganglia were performed in 5 random ganglionic fields for each mouse using confocal microscopy and Nis Elements vs. 3.0 as image analysis system. The cholinergic network of longitudinal muscle-myenteric plexus was evaluated in 8 random fields by optical microscopy and the following optical density distribution of AChE by ImageJ vs. 1.14 program. Protein expression of peripherin, nNOS and pAKT was assessed by western blot analysis.
A significant reduction of the ganglionic areas in the myenteric plexus was found in mice 1 (-33%) and 8 W (-18%) postinfection (PI). S-100 β positive glial cells increased from 22±3 insham to 36±4 inmice 4 W PI. In the ENS the Hu C/D labelling was significantly decreased at 1 W PI (-50%) and further reduced at 8 W after HSV-1 infection. By performing peripherin staining an irregular distribution and interrupted neurofilaments were found at 1 and 8 W PI whereas protein levels were significantly increased (+42%) at 1 W PI. In longitudinal muscle-myenteric plexus preparations, nNOS protein levels were significantly increased at 1 and 4 W (+56% and +37%, respectively) post IG inoculum, confirming an altered NANC inhibitory pathway. Akt phosphorylation was also significantly increased during viral infection. Furthermore, a reduced staining of acetylcholine esterase-positive neurons, large fibers and small fibers, was evident at 1 and 8 W PI.
These results provide evidence that HSV-1 induced neuronanatomical and neurochemical alterations in the ENS, leading to a chronic neuropathy. HSV-1 infection of ENS appears to be a suitable animal model for studying the role of ENS in the onset of GIMDs.
1. Gesser RM, Koo SC (1996) J Viriol 70: 4097-102
2. Brun P et al. (2010) Gastroenterology 138:1790-801