Intranerval TMEV infection (N. hypoglossus; N. ischiadicus)

Chapter 3 Introduction

3.3 Different routes of experimental TMEV infection in mice

3.3.4. Intranerval TMEV infection (N. hypoglossus; N. ischiadicus)

To substantiate the thesis that TMEV travels along motoric nerve fibres, a direct injection of a high-neurovirulent TMEV strain (GDVII) into the N. hypoglossus was performed. Infected mice showed paralysis of the tongue or forelimbs but no combination of both (VILLARREAL et al., 2006). However, viral infections do not only utilize the PNS for trafficking and spread, they are also able to cause direct damage to the nerves (PFISTER, 1999; SUSALKA and PFISTER, 2000). There are various virus-induced demyelinating diseases primarily affecting the PNS, like Marek’s disease in chicken or some subtypes of Guillain-Barré syndrome (GBS) in humans (ARSTILA et al., 1971; DRESCHER and TRACY, 2007b; HUGHES et al., 2016; LAMPERT et al., 1977; LAMPERT, 1978). After direct injection of the low-neurovirulent strain (DA) into the sciatic nerves of mice, TMEV was detected within the nerval tissue and replicating virus could also be harvested. Moreover, an infiltration of macrophages and T cells as well as a demyelination of nerve fibres was found. Virus spread to the SC occurred one week after infection (DRESCHER and TRACY, 2007b).

INTRODUCTION

17 3.3.5. Intra-footpad TMEV infection

Another infection route used for investigating axonal transport and spread of high-neurovirulent TMEV (GDVII strain) is intra-footpad (i.f.) inoculation. Infected mice showed paralysis of the inoculated leg and subsequent involvement of the contralateral side (VILLARREAL et al., 2006). Virus spread was observed from the footpad to the ipsilateral sciatic nerve, the inferior SC, followed by superior SC. This findings indicate that TMEV travels alongside the nerve fibres innervating musculature and skin of the footpad (MARTINAT et al., 1999). Obtained results from i.f. injection coincided with those obtained after intramuscular injection into the gastrocnemius muscle, also entailing paralysis of forelimbs. SC lesions characterized by inflammation (meningomyelitis; perivascular cuffs) as well as neuronal degeneration (necrosis) could be observed. The neuronal loss consistently corresponded to the manifestation of clinical disease in paralyzed animals (VILLARREAL et al., 2006).

3.3.6. Intravenous TMEV infection

Intravenous (i.v.) infection of mice with high-neurovirulent TMEV strains (GDVII) in CBA mice resulted in a high number of mice showing paralysis of hind- and/or forelimbs.

The distribution of paralysis was correlated with the development of SC lesions consisting of inflammation and neuronal degeneration (VILLARREAL et al., 2006). I.v.

injection of the DA strain of TMEV has been shown to stimulate an antiviral immune response in SJL mice, but does not efficiently lead to an infection of the CNS (TSUNODA et al., 2005; TSUNODA et al., 2007).

3.3.7. Intracerebral TMEV infection

I.c. TMEV infection of young adult, susceptible mice with low-neurovirulent TMEV strains leads to the development of TMEV-IDD (GERHAUSER et al., 2019). TMEV strains of the GDVII group cause a fatal encephalitis with lysis of neurons following i.c.

infection regardless of the mouse strain (AUBERT and BRAHIC, 1995; DAL CANTO and LIPTON, 1980; THEILER and GARD, 1940). TMEV strains of the TO group cause a biphasic disease following i.c. infection, consisting of an acute polioencephalomyelitis which is followed by chronic progressive inflammation and demyelination in the SC, associated with virus persistence (DEPAULA-SILVA et al., 2017; GERHAUSER et al., 2019; LIPTON, 1975; LIU et al., 1967; MECHA et al., 2013;

INTRODUCTION

OLESZAK et al., 2004). Clinical signs during the acute phase are frequently mild and reversible (LIPTON, 1975). The late phase of TMEV-IDD constitutes a well-known animal model for the investigation of the pathogenesis of demyelinating diseases like MS (DAL CANTO et al., 1996; RODRIGUEZ et al., 1987). Mechanisms leading to the development of autoimmune processes against oligodendrocytes in TMEV-IDD include an epitope spread as well as molecular mimicry between viral and myelin epitopes (DONATI, 2020; KATZ-LEVY et al., 2000; MILLER et al., 1997; OLSON and MILLER, 2005). I.c. infection of resistant mouse strain (e.g. B6) results in an acute polioencephalomyelitis including hippocampal lesions with neuronal loss and infiltration of T lymphocytes and macrophages as well as microgliosis (GERHAUSER et al., 2019). However, B6 mice eliminate low-neurovirulent TMEV strains from the CNS approximately until four weeks post infection (LINDSLEY and RODRIGUEZ, 1989;

NJENGA et al., 1997).

3.3.8. Intraspinal TMEV infection

An intraspinal (i.s.) TMEV infection model investigating the early events of virus-induced demyelination in the SC is described using a DA strain of TMEV in SJL mice (DRESCHER and TRACY, 2007a). In this model the authors described the replication of TMEV at the injection site along with a demyelination (DRESCHER and TRACY, 2007a). The data of this study indicate, that the initial phase of polioencephalitis is not mandatory for investigating TMEV-induced demyelinating lesions.

3.4. Hypothesis and Aims

It seems that besides numerous variables (sex, age, virus and mouse strain used), the route of infection has a strong effect on clinical signs, virus spread, neuroinfectivity and organotropism. However, a direct comparison of i.c. and i.s. infection neither in SJL nor other mice has been conducted so far. The hypothesis of this thesis was, that i.s.

TMEV infection represents a suitable model for studying demyelinating SC as well as PN lesions in mice with a susceptible genetic background.

Therefore, the first aim was to directly compare the occurrence and course of SC pathology in susceptible mice after i.c. and i.s. TMEV infection. Special emphasis was given to the time course, inflammatory infiltrates, the emergence of demyelinating

INTRODUCTION

lesions and establishment of virus persistence. During a second experiment, i.s. TMEV infection of resistant B6 mice was implemented. The aim of this second study was to investigate whether direct injection of TMEV into the SC of resistant mice could induce demyelinating lesions comparable to those seen in SJL mice after i.s. infection, by circumventing the missing establishment of virus persistence after i.c. infection.

MANUSCRIPT 1: Comparison of TMEV-induced SC and PN lesions following i.c. and i.s. infections

Chapter 4 Comparison of Theiler's murine encephalomyelitis virus induced spinal cord and peripheral nerve lesions following intracerebral and intraspinal infection

Wen Jin, Eva Leitzen, Sandra Goebbels, Klaus-Armin Nave, Wolfgang Baumgärtner, Florian Hansmann

Abstract

Hallmarks of Theiler’s murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) include spinal cord (SC) inflammation, demyelination and axonal damage occurring approximately 5–8 weeks after classical intracerebral (i.c.) infection.

The aim of this study was to elucidate the consequences of intraspinal (i.s.) TMEV infection and a direct comparison of classical i.c. and intraspinal infection. Swiss Jim Lambert (SJL)-mice were i.s. infected with the BeAn strain of TMEV. Clinical investigations including a scoring system and rotarod analysis were performed on a regular basis. Necropsies were performed at 3, 7, 14, 28 and 63 days post infection (dpi) following i.s. and at 4, 7, 14, 28, 56, 98, 147 and 196 dpi following i.c. infection.

Serial sections of formalin-fixed, paraffin-embedded SC and peripheral nerves (PN) were investigated using hematoxylin and eosin (HE) and immunohistochemistry. I.s.

infected mice developed clinical signs and a deterioration of motor coordination approximately 12 weeks earlier than i.c. infected animals. SC inflammation, demyelination and axonal damage occurred approximately 6 weeks earlier in i.s.

infected animals. Interestingly, i.s. infected mice developed PN lesions, characterized by vacuolation, inflammation, demyelination and axonal damage, which was not seen following i.c. infection. The i.s. infection model offers the advantage of a significantly earlier onset of clinical signs, inflammatory and demyelinating SC lesions and additionally enables the investigation of virus-mediated PN lesions.

Int J Mol Sci. 2019: Oct 16;20(20):5134.

https://www.mdpi.com DOI: 10.3390/ijms20205134.

MANUSCRIPT 2: Virus-triggered spinal cord and peripheral nerve lesions

Chapter 5 Virus-triggered spinal cord demyelination is followed by a peripheral neuropathy resembling features of Guilain-Barré Syndrome

Eva Leitzen, Barbara Raddatz, Wen Jin, Sandra Goebbels, Klaus-Amin Nave, Wolfgang Baumgärtner, Florian Hansmann

Abstract

Theiler’s murine encephalomyelitis virus (TMEV)-induces a demyelinating disease in the spinal cord (SC) of susceptible but not in resistant (B6) mouse strains. The aim of the present study was to induce SC demyelination and a peripheral neuropathy in resistant mice by switching the infection site from cerebrum to SC. B6 mice were intraspinally inoculated with TMEV. Infected mice showed clinical signs starting at 7 days post infection (dpi). Histopathology revealed a mononuclear myelitis, centred on the injection site at 3 dpi with subsequent antero- and retrograde spread, accompanied by demyelination and axonal damage within the SC. Virus protein was detected in the SC at all time points. SC inflammation decreased until the end of the investigation period (28 dpi). Concurrent with the amelioration of SC inflammation, the emergence of a peripheral neuropathy, characterized by axonal damage, demyelination and macrophage infiltration, contributing to persistent clinical sings, was observed.

Intraspinal TMEV infection of resistant mice induced inflammation, demyelination and delayed viral clearance in the spinal cord and more interestingly, subsequent, virus-triggered inflammation and degeneration within the PN associated with dramatic and progressive clinical signs. The lesions observed in the PN resemble important features of Guillain-Barré syndrome, especially of acute motor/motor-sensory axonal forms.

Sci Rep. 2019 Mar 14;9(1):4588.

www.nature.com

DOI: 10.1038/s41598-019-40964-1.

DISCUSSION

25 Chapter 6 Discussion

6.1 Intracerebral versus intraspinal TMEV-infection of susceptible mice Amongst various routes of experimental TMEV infection, i.c. infection of susceptible mouse strains with TO virus strains represents an excellent animal model for studying virus-induced demyelinating diseases of the CNS (DAL CANTO et al., 1996; LIPTON and DAL CANTO, 1979b; MECHA et al., 2013; TSUNODA and FUJINAMI, 1996;

TSUNODA et al., 2016). The present study was conducted to test the hypothesis whether i.s. infection leads to the establishment of virus persistence and a subsequent demyelination within the SC of susceptible mice, comparable to that seen in TMEV-IDD following i.c. infection. Previous studies showed that the thoracic SC is the most affected SC segment compared to the cervical and lumbar segments following i.c.

infection (LIPTON and DAL CANTO, 1979a). Therefore, thoracic SC was selected for direct i.s. TMEV infection in mice. A direct injection into thoracic SC was thought to result in an earlier onset of lesions due to the circumvention of the initial phase of encephalitis after i.c. injection. Previous studies investigating different TMEV infection localizations including footpad, skeletal muscle and peripheral nerve demonstrated a virus spread, especially via the PNS to the SC (DRESCHER and TRACY, 2007b;

MARTINAT et al., 1999; RUSTIGIAN and PAPPENHEIMER, 1949; VILLARREAL et al., 2006). Therefore, it was hypothesized, that switching the injection site from cerebrum to SC will result in a virus spread from the SC to the PNS.

In the present study i.s. TMEV infection induced similar clinical signs as described for mice in TMEV-IDD, characterized by flaccid limb paralysis and an impairment of motor coordination. SC lesions consisted of mononuclear inflammation and demyelination, consistent with morphological findings during TMEV-IDD following i.c. infection.

However, the time span between TMEV infection and the occurrence of clinical signs as well as histopathological lesions including demyelination was significantly shorter following i.s. infection compared to i.c. infection. In detail, clinical investigation of i.s.

infected mice revealed a significantly elevated clinical score and a deterioration of motor performance starting at 11 dpi and 14 dpi, respectively. Comparable findings in TMEV-IDD following i.c. infection occurred approximately 12 weeks later (starting at 98 dpi). Accordingly, an approximately 6 weeks shorter (14 dpi vs. 56 dpi) time span before onset of SC inflammation, demyelination and axonal damage was observed in i.s. compared to i.c. infected mice. This significantly shorter time span was most likely

DISCUSSION

an immediate consequence of bypassing the acute phase of polioencephalitis occurring in mice following i.c. TMEV infection. Inflammation and demyelination disseminated from the injection site to the cervical as well as lumbar SC segments, contrasting the unidirectional, caudally orientated, propagation in mice following i.c.

infection. The distinct primary lesion site within SC following i.s. infection constitutes an important benefit, since primary and secondary demyelinating lesions can be distinguished, and the velocity and direction of viral spread can be tracked. The shortened time period between infection and occurrence of inflammation and demyelination in the SC of i.s. TMEV infected mice reduces time and monetary investments, and it also enables the investigation of more progressed lesion with lower risk of interfering with age-related degenerative changes in susceptible SJL mice including spontaneous myopathy or lymphoma (CHOW and HO, 1988; WELLER et al., 1997). TMEV-IDD following i.c. infection is mainly characterized by demyelinating and inflammatory WM lesions without significant GM involvement (DAL CANTO et al., 1996;

TSUNODA et al., 2001). After i.s. infection, in addition to a leukomyelitis a poliomyelitis, starting at 14 dpi, with occasional neuronal degeneration was observed. The poliomyelitis after i.s. infection may reflect the acute polioencephalitis following i.c.

TMEV infection. It is likely that neuronal degeneration has contributed to the early onset of clinical deterioration in this study. Moreover, impairment of motor coordination was also correlated with the emergence of PN lesions. Alterations within PN were characterized by mononuclear infiltrates, axonal damage, demyelination and vacuolation as well as presence of virus protein. Interestingly, following i.c. TMEV infection, significant PN damage has not been observed and virus was only rarely detected within the PNS. The increasing amount of detectable virus protein can be indicative for a continuous virus spread from CNS towards PNS and/or a proliferation of virus within PN. Regarding the mechanism of virus transmission, different ways can be envisaged. First, a transmission by bidirectional axoplasmic flow or second, intracellular spread within infected macrophages, or a combination of both (DRESCHER and TRACY, 2007b; LIPTON et al., 1995). The occurrence of axonal damage may follow different mechanisms: a primary damage to the myelin sheath followed by secondary axonal degeneration (outside-in mechanism), a primary axonal damage followed by secondary demyelination (inside-out mechanism) or a combination of both (KREUTZER et al., 2012; LIBBEY et al., 2014; TSUNODA and FUJINAMI, 2002). Whether inflammatory reactions within PN represent an event

DISCUSSION

related to SC inflammation, or an independent phenomenon as possible result of a compartmentalized immune response between PNS and CNS (NAVARRETE-TALLONI et al., 2010) needs to be unravelled in future studies.

In conclusion it can be noted that i.s. TMEV infection in susceptible SJL mice leads to comparable clinical signs and histopathological lesions as observed in susceptible SJL mice following i.c. infection, with the advantage of a significantly shortened investigation period as well as the additional possibility to investigate subsequent PN lesions.

6.2 Intraspinal TMEV-infection of susceptible mice versus resistant mice It has been shown that the clinical and pathohistological outcome of TMEV infection is variable and depends on the infection route as well as the virus and mouse strain used.

Infection via i.p. route, using the DA strain of TMEV, results in a myositis in numerous mouse strains, while the occurrence of myocarditis is especially seen in C3H mice (GOMEZ et al., 1996; RODRIGUEZ et al., 1986). After i.c. infection using a low virulent strain of TMEV, susceptible mouse strains with subsequent emergence of TMEV-IDD, and resistant strains being able to clear the virus from the CNS, can be distinguished.

Within the previous study, i.s. TMEV-infection was introduced as an alternative way to induce SC lesions, similar to those in TMEV-IDD in susceptible mouse strains. The main question of the second study was whether a switch of injection site (i.c.  i.s.) would affect the clinical and pathohistological outcome in mice on a resistant genetic background (B6). Special emphasis was given to the development of inflammatory and demyelinating lesions, potential establishment of virus persistence and the emergence of a demyelinating peripheral neuropathy after i.s. TMEV-infection. I.s. infected B6 like SJL mice developed clinical signs starting at 11 dpi and a deterioration of rotarod performance starting at 7 dpi (SJL: 14 dpi). SC lesions were predominantly located within the ventral aspects of SC WM, comparable to the obtained results in SJL mice after i.s. as well as i.c. infection. Moreover, phenotyping of inflammatory cells revealed a predominance of CD107b-positive microglia/macrophages and CD3-positive T cells in inflammatory infiltrates within meninges and around vessels. CD45R-positive B cells were also involved, but to a lesser extend compared to i.s. infected SJL mice. Virus protein was detected in the SC of both, i.s. infected SJL and B6 mice. According to the literature, B6 mice are able to eliminate TMEV from the CNS within two to three weeks

DISCUSSION

after i.c. infection (GETTS et al., 2010; OLESZAK et al., 2004). In this context, it is important to note that at the end of the investigation period of this study (28 dpi), TMEV was occasionally detected within the SC of resistant B6 mice. The presence of virus protein within the CNS four weeks after infection could be indicative of a delayed viral clearance. However, the establishment of a virus persistence in B6 mice was not investigated in this study. It’s already known that, due to the elimination of TMEV, i.c.

infected B6 mice do not develop demyelinating lesions neither in the CNS nor the PNS (JAFARI et al., 2012; OLESZAK et al., 2004). However, following i.s. infection of B6 mice, demyelinating lesions were found not only in SC but also in PN. Comparable to i.s. infected SJL mice, B6 mice exhibited a vacuolation of nerve fibers as well as a hypercellularity within PN, characterized by mononuclear infiltrates starting at 14 dpi, in both, SJL and B6. Moreover, a demyelination and the occurrence of axonal damage, starting at 14 dpi (B6) or 28 dpi (SJL) was noticed. This accelerated onset of clinical deterioration and the emergence of demyelinating lesions could be on the one hand a result of a more extensive collateral tissue damage due to the more effective immune response resulting in virus clearance in B6 mice. On the other hand it could be a hint towards a compartmentalization of immune responses between brain and SC, as already described in literature for traumatic injuries in B6 CNS (SCHNELL et al., 1999a;

SCHNELL et al., 1999b).

One important difference between both mouse strains was the number of TMEV-positive cells within the PN. Virus protein within the PN was detected after 7 dpi (SJL and B6), demonstrating that TMEV is able to spread anterograde from SC to the PNS.

The observed time frame is in accordance with the observed results after intranerval (ischiadic nerve) infection, where TMEV travelled retrograde and could be harvested from SC after 7 dpi (DRESCHER and TRACY, 2007b). Although different routes of virus spread have been discussed for TMEV (see chapter 3.3), direct axonal transport and/or virus spread via infected macrophages seem to be the most obvious explanation in this model. However, further studies are needed to elucidate the cellular tropism and route of virus spread within PN. The number of TMEV-infected cells in SJL mice increased over time, indicating an ongoing spread to PN or a viral proliferation within PN. Myelin injury might therefore be a nonspecific consequence of an antiviral immune response as a kind of “bystander effect” (LIPTON and DAL CANTO, 1979a).

In B6 mice, however, only single TMEV positive fibers in single animals were detected at 7 and 14 dpi. Therefore, the underlying mechanism that triggers demyelination is

DISCUSSION

probably not strongly depending on the presence of virus within the PN. Therefore, it could also be the result of autoimmune reactions (e.g. epitope spreading, molecular mimicry), comparable to those responsible for demyelination within the SC after i.c.

infection (MILLER et al., 2001; OLSON et al., 2001).

6.3 Conclusion

In conclusion, after i.s. TMEV infection, susceptible SJL and resistant B6 mice showed a clinical deterioration. Moreover, the observed clinical signs correlated with inflammatory, degenerative and demyelinating lesions within the SC, comparable to those seen in TMEV-IDD after i.c. infection of SJL mice, but with the advantage of a markedly shortened time span between infection and clinical onset as well as histopathological lesions. Moreover, the i.s. infection route enables the precise spatial and temporal investigation of virus spread and lesion sites. An addition advantage is that with this model also mice on a resistant B6 background can be used for studying inflammatory and demyelinating SC lesions. However, an establishment of virus persistence was only detected in i.s. infected SJL mice. Interestingly, after i.s. infection, inflammation, degeneration and demyelination were not restricted to the CNS but also occurred in PN of both, SJL and B6 mice. This peripheral neuropathy following i.s.

infection resembles important features of subforms of GBS, indicating its potential as a feasible model for human PNS diseases, making the i.s. TMEV-model an interesting alternative to classical i.c. infection. The obtained results once again clearly illustrate the profound impact of the injection site towards the clinical and pathohistological outcome following TMEV infection.

REFERENCES

31 Chapter 7 References

Allnoch, L., Baumgärtner, W., Hansmann, F. (2019): Impact of astrocyte depletion upon inflammation and demyelination in a murine animal model of multiple sclerosis. Int. J. Mol. Sci. 20, 3922 Anderson, R., Harting, E., Frey, M. S., Leibowitz, J. L., Miranda, R. C. (2000): Theiler's murine

Im Dokument Intraspinal Infection with Theiler’s Murine Encephalomyelitis Virus in Resistant and Susceptible Mouse Strains (Seite 30-0)