Brain Research 868 (2000) 259–267www.elsevier.com/ locate /bres

Research report

Theiler’s murine encephalomyelitis virus induces rapid necrosis anddelayed apoptosis in myelinated mouse cerebellar explant cultures

a ,1,2 a ,2 c b ,3Rachel Anderson , Eric Harting , Miles S. Frey , Julian L. Leibowitz ,a ,*,3Rajesh C. Miranda

aDepartment of Human Anatomy, Texas A&M University Health Science Center, 228 Reynolds Medical Bldg., College Station, TX 77843-1114, USAbDepartment of Pathology and Laboratory Medicine, Texas A&M University Health Science Center, College Station, TX 77843-1114, USA

cDepartment of Veterinary Pathobiology, Texas Veterinary Medical Center, Texas A&M University, College Station, TX 77843, USA

Accepted 28 March 2000

Abstract

Infection with the Daniel strain of Theiler’s murine encephalomyelitis (TMEV-DA) virus induces persistent demyelinating lesions inmice and serves as a model for multiple sclerosis. During the acute phase of the disease, however, viral infection leads to cell death invivo. Viral-induced death may result directly from viral infection of neural cells, or indirectly, by activation of the immune system. Toexamine the direct effects of TMEV infection on neural cells, myelinated explant cultures of the murine cerebellum were infected with

510 pfu of TMEV-DA for periods ranging from 1 to 72 h. Our results indicate that TMEV-DA replicates in cultured neural tissue. Initially,viral antigen is localized to a few isolated neural cells. However, within 72 h antigen was observed in multiple foci that included damagedcells and extracellular debris. Viral infection led to a rapid and cyclical induction of necrosis with a time period that was consistent withthe lytic phase of the viral life-cycle. Simultaneously, we observed an increase in apoptosis 48 h post-infection. Electron micrographicanalysis indicated that viral-infected cultures contained cells with fragmented nuclei and condensed cytoplasm, characteristic of apoptosis.The localization of apoptosis to the cerebellar granule cell layer, identified these cells as presumptive granule neurons. Viral infection,however, did not lead to myelin damage, though damaged axons were visible in TMEV-infected cultures. These results suggest that duringthe acute phase of infection, TMEV targets neural cells for apoptosis without directly disrupting myelin. Myelin damage may thereforeresult from the activation of the immune system. 2000 Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Degenerative disease: other

Keywords: Lactate dehydrogenase; Apoptosis; Necrosis; Mouse; Cerebellum; Explant; Myelin; Picornavirus; TMEV

1. Introduction brain and spinal cord. Viral replication is prominent inneurons, but can also be detected in astrocytes and

The Daniel (DA) strain of Theiler’s murine en- microglial cells [3]. The second phase of the disease iscephalomyelitis virus (TMEV), a picornavirus, induces a characterized by primary demyelination and extensivebiphasic disease after intracerebral inoculation of adult inflammatory infiltrates in the white matter, and thusmice [19]. The initial phase of the disease is characterized provides a good animal model for multiple sclerosis.by a transient and mild neuronal poliomyelitis with virus The mechanisms underlying demyelination in this modelreplication occurring primarily in the gray matter of the are incompletely understood. In the natural infection,

TMEV is thought to gain access to the CNS via replicationin the cells that form the blood–brain barrier [37,40] or in

*Corresponding author. Tel.: 11-979-862-3418; fax: 11-979-845- virus-infected macrophages [9]. In the late demyelinating0790. phase of the disease the virus has been identified in

E-mail address: rmiranda@tamu.edu (R.C. Miranda)1 astrocytes, oligodendrocytes and macrophages within theCurrent address: University of Texas, Houston, TX, USA.2 CNS [4]. Pharmacological immunosuppression decreasesThese authors made equal contributions to the research.3These authors were co-P.I.s on this research project. the extent and severity of demyelination [20], suggesting

0006-8993/00/$ – see front matter 2000 Elsevier Science B.V. All rights reserved.PI I : S0006-8993( 00 )02338-6

260 R. Anderson et al. / Brain Research 868 (2000) 259 –267

that demyelination is linked to the host immune response. termined by plaque assay on monolayers of L2 cells asDirect cytotoxic effects of virus infection of neural tissue described previously [28].may also contribute to demyelination. Support for thismechanism includes the detection of viral antigens within 2.2. Miceoligodendrocytes [27], a correlation between the presenceof viral RNA in white matter and demyelinating lesions Timed pregnant mice (C3H-HEN) were purchased from[7], the presence of demyelinating lesions during infections Harlan (TX), maintained in an NIH approved facility on aof nude mice [30], and similar observations during in- 14:10-h light–dark cycle and provided with food and waterfections of mice, in which functional CD4 and CD8 T-cell ad libitum. Post-natal day 1 was defined as the day ofpopulations have been eliminated by genetic knockouts birth. Mouse cerebellar cultures were established from[25,29]. mice on post-natal day 2.

While the pathogenesis of chronic TMEV infection hasbeen studied extensively, the acute phase of infection is not

2.3. Organotypic explant cultures of the cerebellumas well understood. Over the past few years many viruses,including members of the picornavirus family, have been

Murine explant cultures of the developing cerebellumfound to induce apoptosis [34]. This effect is thought towere established from C3H-HEN mice on post-natal day 2contribute to the acute cytopathogenic effects of theseaccording to previously published protocols [22,23]. Briefl-viruses [11,33]. The BeAn strain of TMEV inducesy, mouse brains were dissected out under sterile conditionsapoptosis in a simian kidney-derived cell line (BSC-1) [15]and separated from meningeal tissue. Cerebella weresuggesting a possible role for apoptosis in the pathogenesismicrodissected out and sectioned into 360-mm sagittalof TMEV-induced disease. Recent evidence indicatessections using a brain slicer [35]. Cultures were establishedinfection of mice with TMEV-DA leads to the induction ofon collagen-coated coverslips and maintained in roller tubeapoptosis in neurons throughout the brain during the acuteassemblies in the presence of 0.5 ml culture mediumphase of the disease [36]. The induction of apoptosis couldcontaining BME and Hank’s BSS, 25% gelded horsebe either the result of direct infection of neural cells by theserum (JRH) and 0.65% glucose. Explant cultures werevirus or an indirect result of immune system activationincubated at 34.58C in 5% CO in a cell culture roller2following infection.drum (Bellco), rotated at 2 rpm. Cultures were maintainedIn the following studies, we therefore infected maturefor 15 days in vitro until differentiation and myelinationmurine explant cultures of the myelinated cerebellum withwas well established [1,2]. We have previously observeda persistent strain of TMEV (TMEV-DA), to determine ifthat cell death in roller tube explant cultures (measured byTMEV can directly induce apoptosis or necrosis in neuralDNA fragmentation and release of lactate dehydrogenasetissue, independent of effects resulting from viral activa-activity) decreases to virtually undetectable levels by 6tion of the immune system. The cerebellum was selected asdays in culture [8]. On day 16, cultures were exposed toan appropriate model because it myelinates well in vitro 510 pfu of TMEV-DA or control medium for 1 h. Culture[1,2], is an in vivo site of TMEV infection [38] and is amedium was replaced and cultures were maintained for 24,common site for plaque formation in multiple sclerosis48 or 72 h, then processed for immunohistochemical[14].analysis, electron microscopic analysis, virus titration orfor the detection of apoptosis and necrosis.

2. Materials and methods 2.4. Immunohistochemical analysis of TMEV-DAexpression

2.1. Virus and cellsCultures were fixed for 30 min in 4% phosphate-buf-

A small plaque variant of TMEV-DA known as D was fered paraformaldehyde and 2.5% DMSO. After fixation,s

used for all of the experiments in this work. The origin and the cells were washed 3 times in cold Tris-buffered salinegrowth of this virus has been described previously [26]. D (TBS, pH 7.4), then processed for the detection of TMEV-s

produces minimal acute encephalomyelitis during the first DA antigen. Briefly, cultures were blocked with a solution10 days following intracerebral inoculation followed by containing 5% normal donkey serum, 1% BSA, and 0.3%prominent inflammatory demyelination by 45 days post- Triton X-100 in TBS. Cultures were incubated with ainfection. To assay the amount of infectious virus in rabbit antisera raised against purified TMEV-DA [27,28] atorganotypic explant cultures, virus was released from the a 1:100 dilution in blocking solution overnight at 48C.cultures by three cycles of freeze–thaw at 2708C followed Binding of the primary antibody was detected using aby 3320-s bursts of sonication (100 W peak envelop biotinylated secondary antibody (donkey anti-goat 1:500,power) in a cup sonicator. After low-speed centrifugation Vector Labs.) conjugated to an avidin–biotin–horseradishto sediment debris, titers of infectious virus were de- peroxidase complex (ABC elite Kit, Vector laboratories)

R. Anderson et al. / Brain Research 868 (2000) 259 –267 261

and a chromogenic substrate (Ni-diaminobenzidine, Vector acetate followed by lead citrate [31] and examined with aLabs). transmission electron microscope (Zeiss IOC, Carl Zeiss,

NY).2.5. Lactate dehydrogenase (LDH) assay

2.8. Data analysisCulture-conditioned medium was obtained from control

or TMEV-DA-infected cerebellar explant cultures. LDHChanges in viral load, LDH release and DNA frag-

activity released into the culture medium (a measure of cellmentation were analyzed with a standard statistical pack-

membrane lysis due to necrotic cell death) was assayedage (SPSS) using analysis of variance (ANOVAs) followed

using a Cytotoxicity Detection Kit (Boehringer Mann-by Fisher’s post-hoc tests. Sample sizes for each data point

heim/Roche Pharmaceuticals) according to the manufac-ranged from three to seven and represent two to three

turer’s instructions. Briefly, culture-conditioned media wasindependent experimental replications. Levels of statistical

rendered cell-free by centrifugation at 2503g. One hun-significance were set at P#0.05 and mean values were

dred ml of conditioned medium from each sample wereplotted, with standard error of the mean (S.E.M.) indicated

added to a 96-well microtiter plate. LDH activity wason graphs by error bars. Mean difference measures that

measured by a redox reaction that couples the oxidation ofexceed statistical significance are indicated in the graphs

lactate to pyruvate with the reduction of the chromogenicby asterisks.

substrate iodotetrazolium chloride to a colored formazansalt, using NADH as the electron transfer agent and NADHdiaphorase as the catalyst. Absorbence of samples wasmeasured at 490 nm against a reference wavelength of 630 3. Resultsnm using a microtiter plate reader (ELx808, BiotekInstruments). LDH levels were quantified against a panel 3.1. Viral content in cerebellar culturesof LDH standards ranging in concentration from 1 mM to0.001 nM. Each mature cerebellar culture, previously maintained

5for 15 days in vitro, was infected with a total of 10 pfu of2.6. Cell death (apoptosis) ELISA TMEV-DA for 1 h. Following this period of infection,

virus-containing culture medium was aspirated and re-Apoptosis in cortical explants was quantified using a cell placed with fresh, non-virus containing medium. The

death ELISA kit (Boehringer Mannheim/Roche Pharma- amount of infectious virus in each cerebellar explantceuticals) according to the manufacturer’s protocols. culture was measured at 1, 24, 48 or 72 h post-infection.

2Briefly, cells were lysed with an incubation buffer and At 1 h post-infection, a mean viral titer of 2.7310small molecular weight, cytoplasmic histone-bound DNA pfu/culture was recovered from infected cerebellar cul-fragments were separated from intact nuclei by centrifuga- tures (Fig. 1). At the end of 24 h, there was a significant

4tion at 20,0003g for 15 min. The supernatant was exposed increase in the mean viral titer to 1.1310 pfu /culture.to anti-histone antibody-coated 96-well plates for 90 min There was no significant change in the amount of virusand washed. Plates were subsequently exposed to anti- recovered from 24 to 72 h post-infection.DNA antibody conjugated to peroxidase for 90 min, thenwashed. Finally, the plates were exposed to a chromagenicsubstrate (ABTS; 2,29-azino-di-[3-ethylbenzthiazoline sul-fonate]) for colorimetric quantification at 405 nm using amicrotiter plate reader (Elx808, Biotek Instruments).

2.7. Electron microscopic analysis

Cultures were prepared for electron microscopic analysisusing a modification of previously published protocols[18]. Cultures were fixed in Karnovsky’s fixative overnightat 48C, washed in 0.1 M sodium cacodylate buffer, post-fixed in 1% OsO in 0.1 M sodium cacodylate buffer for 24

h and washed again. Cultures were then immersed insaturated uranyl acetate in H O, overnight at 48C, sub-2

sequently dehydrated with ethanol followed by 100%Fig. 1. Alterations in viral yield per culture between 1 and 72 h post-

propylene oxide and embedded in epon araldite. Thin 5infection with 10 pfu of TMEV-DA. Our results indicate that there was asections (between 60 and 90 nm) were then cut, mounted statistically significant increase in viral yield per culture, between 1 andon copper grids, then re-stained with saturated uranyl 24 h post-infection. Viral yield remained constant thereafter.

262 R. Anderson et al. / Brain Research 868 (2000) 259 –267

3.2. Immunohistochemical analysis of viral infection media was used as an index of cell lysis and hence,necrosis. Control, mock-infected cerebellar explant cul-

Immunohistochemical analysis indicated that viral an- tures maintained for 15 days in vitro, exhibited low levelstigen was detected in the cytoplasm of single, widely of LDH release that was below the sensitivity of the assay.dispersed cells at 24 h post-infection (Fig. 2b,d versus At 24 h post-infection, there was a substantial and statisti-controls in 2a). However, at 72 h post-infection, viral cally significant increase in LDH release into culture mediaantigen was observed in clusters of cells (Fig. 2c). Im- (Fig. 3). This was followed at 48 h, by a significant, 6-foldmunopositive clusters included apparently healthy cells decline in released LDH. At 72 h post-infection, released(Fig. 2d) as well as pyknotic cells and other cellular LDH levels were again increased, so that they were notdetritus. Infected pyknotic cells demonstrated the accumu- different from the levels observed at 24 h post-infection.lation of cytoplasmic vacuoles (Fig. 2e, arrows) as well asmulti-lobular nuclei (Fig. 2f,g, arrows), a feature charac- 3.4. TMEV-DA induces a delayed increase in apoptosisteristic of apoptosis. in cerebellar explant cultures

3.3. TMEV-DA induces necrosis in cerebellar explant Levels of small molecular weight, cytoplasmic histone-cultures associated DNA were measured by sandwich ELISA assay

and used as a measure of apoptosis (Fig. 4). Low levels ofLactate dehydrogenase activity released into culture apoptosis were observed in mature, post-mitotic control

Fig. 2. Immunohistochemical localization of TMEV-DA to infected explant cultures of the developing cerebellum. Cerebellar explants were cultured for 15days in vitro, then infected with TMEV-DA. Low magnification photomicrographs indicated that mock-infected control cultures (a) demonstrated noTMEV-DA immunoreactivity. At the end of 24 h post-infection (b) TMEV-DA was localized to single cells within the explant, while at 72 h post-infection(c), immunoreactivity was observed within clusters of cells. High magnification photomicrographs indicated that infected cells appeared intact at 24 hpost-infection (d). However, at 72 h post-infection, antigen-presenting cells exhibited vacuolization (e, arrows) and fragmented nuclei (f,g, arrows). Scalebars: (a–c) 60 mm, (d–f) 10 mm.

R. Anderson et al. / Brain Research 868 (2000) 259 –267 263

in vitro, then mock-infected and analyzed 72 h later.Control cerebellar explants contain healthy neuronal somawith well-defined organelles (nuclei, mitochondria, endo-plasmic reticulum and ribosomal complexes) and processes(Fig. 5A,B). Myelinated axons are observed throughout thecorpus of the explant (Fig. 6A,B). In contrast, TMEV-DAinfection of cerebellar explant cultures lead to substantialchanges in morphology by 72 h post-infection. A numberof cells appeared to have undergone cytoplasmic andnuclear fragmentation (Fig. 5C,D). In these cells, cyto-plasmic organelles appear to have been lost. The cyto-plasm contained electron-dense material surroundingmultiple vacuoles. Nuclei of these cells were irregular andfragmented into multiple compartments. Chromatin in eachof these nuclear compartments was condensed, especiallyat the boundary of the nuclear envelope. In TMEV-DAFig. 3. Changes in LDH released into culture medium (a measure ofinfected cultures, we observed no changes in myelinnecrotic cell death) as a function of time following infection. Mock-

infected cultures exhibited virtually undetectable levels of LDH release at laminae surrounding axons (Fig. 6C,D). However, axonsthe end of 15–18 days in culture. In contrast, TMEV-infected cultures were condensed and separated from the surroundingexhibited a significant and cyclical induction of LDH release with a 48-h

myelin by an extracellular space and exhibited degenera-period.tion, leading to the appearance of myelin vacuoles.

cerebellar explant cultures maintained for 15 days in vitro.Cultures examined 24 h post-infection did not contain anincreased number of apoptotic cells relative to uninfected

4. Discussioncontrol cultures. At 48 h, however, there was a statisticallysignificant, 1.8-fold increase in apoptosis in virus-infected

Persistent strains of Theiler’s virus, including the DAcultures as compared to controls. At 72 h post-infection,strain used in this study, induce a chronic demyelinatingthere was a mean increase in apoptosis of 2.5-fold overand inflammatory illness in mice after intracerebral inocu-control mock-infected cultures.lation, and therefore serve as a good model for multiplesclerosis [12,24]. It is thought that the majority of the3.5. Electron microscopic analysis of changes in celldemyelination is immunologically mediated [20]. Thisdeath and myelination in TMEV-DA infected cerebellarchronic phase of TMEV infection is preceded by an earlierexplant culturesstage that is characterized by poliomyelitis accompaniedby brisk virus replication in the gray matter of the brainControl cerebellar explants were maintained for 15 daysand spinal cord. During this phase of the disease, viralantigen is located primarily in neurons, although it can alsobe observed in oligodendrocytes, macrophages and as-trocytes. This acute phase of viral replication in graymatter resolves with the development of an immuneresponse to TMEV.

Recent work demonstrates that following intracerebralchallenge with TMEV substantial apoptosis can be ob-served in vivo throughout the murine brain, most promi-nently in neurons [36]. This apoptotic response wasobserved at all times sampled, although the first data pointreported was at 1 week after challenge. Infection-relatedapoptosis could be directly induced by the virus or,alternatively, be the result of immune system activation.The greatest number of apoptotic cells were detected inareas containing large amounts of viral antigen, rather thanin areas containing perivascular mononuclear infiltrates.

Fig. 4. Changes in DNA fragmentation (apoptosis) as a function of time This pattern suggested that apoptosis of neurons was afollowing infection. The ELISA assay for DNA fragmentation indicated direct effect of infection with TMEV. In vitro studiesthat control, mock-infected cultures exhibited a low but detectable

suggest that TMEV infection triggers apoptosis in abor-apoptosis. In contrast, TMEV-infected cultures exhibited an time-depen-tively infected BSC-1 cells, but not in productively infec-dent increase in apoptosis that was first significantly different from

controls at 48 h post-infection. ted BHK-21 cells [15]. Macrophages have also been shown

264 R. Anderson et al. / Brain Research 868 (2000) 259 –267

Fig. 5. Electron micrographic analysis of mock-infected (A,B) and TMEV-DA-infected (C,D) cerebellar explant cultures at 72 h post-infection.Mock-infected control cultures (A,B) exhibit normal neuronal profiles with euchromatic nuclei, and a complement of cytoplasmic organelles. In contrast,cells in TMEV-infected cultures (C,D, arrows) exhibited nuclear fragmentation (asterisks), chromatin condensation, greater electron-density of thecytoplasm and a lack of well defined cytoplasmic organelles. Arrow-heads (in B and C) indicate neurites en passant. Scale bar, 1.5 mm.

to undergo apoptosis when infected with TMEV [16,17] pfu of virus, TMEV-DA replicates and persists within theand this may play a role in persistence [13]. corpus of the explant for the next 72 h. The level of

In the current studies, we examined the direct effects of infectious virus present in the explant increased during theTMEV infection on neural cells using an explant culture first 24 h and then remained constant thereafter to 72 hmodel of the myelinated cerebellum. The cerebellum post-infection. Immunohistochemical staining for TMEVrepresents a good in vitro model to study demyelinating antigen indicated that single cells were infected at 24 hviruses since it myelinates extensively in culture, and one while clusters of cells were immunopositive for viralneuronal phenotype, the granule cell, represents the domi- antigen at 72 h. This suggests that viral replication andnant cell type both in vivo and in vitro [1,2]. Furthermore, secondary spread of the infection did occur over the 72-hthe laminar organization and temporal patterns of explant time period. The replication of TMEV and the spread ofdevelopment are similar to that observed in vivo. Finally, this virus throughout the explant are unlikely to bethe cerebellum is an in vivo site of TMEV infection [38] associated with a renewed induction of cell cycle /neuro-and is a common site for plaque formation in multiple genesis, since picornaviruses are known to inhibit mitosissclerosis [14]. After 15 days in culture, cerebellar explants and DNA synthesis [21].are largely post-mitotic and differentiated. Cerebellar explant cultures exposed to TMEV-DA un-

5Our results indicate that following infection with 10 dergo a rapid necrosis and a more delayed induction of

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Fig. 6. Electron micrographic analysis of myelination in mock-infected control (A,B) and TMEV-DA-treated (C,D) cerebellar explant cultures.Mock-infected control cultures (A,B) exhibited extensive myelination (arrows). Myelinated axons exhibited well-defined cytoskeletal elements andorganelles such as mitochondria. At 72 h post-infection, TMEV-DA infected cultures also exhibited extensive myelination (C,D, arrows). However axonswithin the myelin sheets (asterisks) appeared degenerated and demonstrated a loss of cytoskeleton and mitochondria. Scale bars: (A,B) 1.7 mm; (C,D) 1.2mm.

apoptosis over 72 h. At 24 h following TMEV-DA induction of necrosis, TMEV-DA-induced apoptosis with ainfection there was a significant release of LDH from the delayed time course. An increase in apoptosis over controlcultures, indicating a rapid compromise of plasma mem- levels was not detected until 48 h post-infection, andbrane integrity and hence necrotic cell death. By 48 h continued to be detected over the next 24 h. Immuno-post-infection, however, there was a reduction in LDH histochemical studies of viral antigen expression demon-release followed by a re-induction of necrosis at 72 h strated viral antigen in cells clearly undergoing apoptosis.post-infection. Though it is unlikely that synchronization Antigen was detected in both small dense cellular frag-of infection was achieved, a 24–36-h cycle of virus ments and in cells with fragmented nuclei, indicating thatreplication, necrosis, and release of TMEV-DA followed at least some TMEV-infected cells underwent apoptosis.by a second cycle of infection and lytic activity would be Electron microscopic analysis indicated that TMEV-infec-consistent with these observations. ted cultures contained apoptotic cells in virtually every

Apoptosis in cerebellar explant cultures was character- section examined. These apoptotic cells were primarilyized by fragmentation of DNA into small histone-associ- observed in the granular neuron layers of the cerebellarated cytoplasmic fragments. In contrast to the rapid explants, suggesting that many of these dying cells may

266 R. Anderson et al. / Brain Research 868 (2000) 259 –267

have been neurons. No apoptotic cells that were un- Referencesequivocally oligodendrocytes, could be identified. Myeli-

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