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DA strain of Theiler's murine encephalomyelitis virus induces demyelination in nude mice.

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BRIEF COMMUNICATIONS AND CASE REPORTS
DA Strain of
Theiler's Murine
Encephalomyelitis Virus
Induces Demyelination
in Nude Mice
Raymond P. Roos, MD,' and Robert Wollmann, MDI
DA strain of Theiler's murine encephalomyelitis virus
produces a chronic, progressive demyelinating disease
in mice that resembles multiple sclerosis. An immunopathological mechanism for demyelination has
been postulated, because there is a brisk immune response with low virus titers at the time of demyelination
and because immunosuppression lessens the degree of
demyelination. We inoculated Nude mice with DA virus
to clarify the role of immune-mediated dernyelination.
Animals became paralyzed 3 weeks to 2 months after
inoculation, usually dying within a week of appearance
of signs. Demyelinated foci were present in the spinal
cords, with evidence of degenerating myelin around intact axons as well as completely demyelinated, naked
axons. Occasional macrophages were present, but none
was seen actively stripping intact myelin lamellae. These
results suggest that DA virus lytic infection, without a
contribution from the T lymphocyte immune system, is
sufficient to produce demyelination. It is likely that DA
virus demyelination has varying mechanisms that may
be active at different times.
Roos RP, Wollmann R: DA strain of Theiler's
murine encephalomyelitis virus induces
demyelination in Nude mice.
Ann Neurol 151494-499, 1984
The DA strain of Theiler's murine encephalomyelitis
virus (TMEV) produces a late, chronic, persistent demyelinating infection of mice that pathologically resembles multiple sclerosis [b, 7). There is controversy
concerning whether viral oligodendrocyte lysis o r immunopathological factors produce D A demyelination.
In this study we examined the effects of DA virus
infection in N u d e mice to elucidate the role of immunopathological mechanisms in the demyelinating
disease; because of their lack of a normal thymus,
Nude mice have been used to delineate the contribution of the immune system in viral-induced models of
From the Departments of *Neurology and tpathology, University of
Chicago Medical Center, Chicago, IL 60637.
Received July 19, 1983, and in revised form Oct 6. Accepted for
publication Oct 8, 1983.
Address reprint requests to Dr Roos.
494
demyelination [4].W e found evidence of chronic demyelination without associated inflammatory infiltrates.
This study indicates that D A virus infection, without a
contribution from the T lymphocyte immune system, is
sufficient to produce demyelination.
Methods
Three groups, totaling 3 1 animals, of 4- to 6-week-old Nude
Balb/c mice (Harlan Sprague-Dawley, Inc.) were inoculated
on separate occasions intracerebrally with serial 10-fold dilutions (10" to 10-j) of a stock containing 6.5 x lo6 plaque
forming units per milliliter of DA virus that had been passed
nine times in BHK-21 cells. Sixteen normal Balbic mice
(Harlan Sprague-Dawley, Inc.) were inoculated with serial
10-fold dilutions ( l o - ' to lo-') of the same virus stock.
Details regarding the growth of the virus and its assay have
been described previously I1 11. The undiluted virus stock
produces demyelination in 100% of 3-week-old SJUJ mite
within 5 weeks of inoculation. When the Nude mice became
ill or moribund, they were perfused with 45F paraformaldehyde under ether anesthesia. The brain and spinal cord
were removed and placed in the paraformaldehyde. After
fixation brain sections were embedded in paraffin, and histological sections were stained with H&E. Spinal cord sections
were embedded in Epon, and semithin sections were stained
with 0.1% toluidine blue. Ultrathin sections of demyelinated
areas were stained with uranyl acetate and lead citrate and
examined with a Philips 201 electron microscope.
An enzyme-linked immunosorbent assay (ELISA)and immunoblotting assay were used for the detection of anti-DA
antibody. In brief, the ELISA involved overnight incubation
at 4°C of purified DA virus onto microtiter plates. Antibody
was then overlaid, followed by washing and then an overlay
with horseradish peroxidase conjugated anti-mouse immunoglobulin G. After further washing, the plates were developed
with 0.012% hydrogen peroxide in 0.1 M citrate (pH, 4.:7)
supplemented with 1 mdml of ortho-phenylenediamine. The
extent of the reaction was determined by measuring the allsorbency of the samples at 430 nm in a Dynatech (Alexandria, Virginia) Microelisa Autoreader, model MR580. The
immunoblotting assay basically followed published techniques [ 5 ] and used purified DA viral polypeptides as the
electrophoresed antigen.
Results
Infected N u d e animals began to show weakness of
their hind legs from 2.5 to 8 weeks after inoculation,
with lowest dilutions causing signs earliest. Mice
tended to develop a paraplegia, although at times three
o r four extremities became paralyzed. Animals usually
became thin and died within a week of showing signs if
they were not killed earlier. No animal lived over 2.5
months after inoculation. Dying animals had no detectable antibody against TMEV, as measured by ELISA
and imrnunoblotting (data not shown).
T h e brains of most of the N u d e mice showed microgliosis, especially in the lower brainstem, at times
with necrotic foci. No lymphocytic infiltrates were observed. A variable number of demyelinated foci involv-
Fig I. Focus of demyelination (XKOWS) in ventral column of
spinal cord of Nude mouse 1 month follozuing inoculation. (Toluidine blue-stained semithin section of Epon-embedded tissue;
x 275 before 5% reduction.)
ing the white matter were found in the spinal cords of 7
of 18 mice that were killed 1 to 2 months after inoculation (Fig 1). Naked axons could be seen in these areas,
indicating a primary demyelination (Fig 2). Large axons
occasionally showed relatively thin loops of myelin.
The anterior horn cells were usually normal in appearance except for vacuolation of occasional neurons.
O n electron microscopic examination the white matter lesions were seen to consist of degenerating myelin
with splitting of lamellae around intact axons (Fig 3), as
well as completely demyelinated, naked axons (Fig 4).
Although a few macrophages were present, none was
found actively removing intact myelin lamellae. There
was a paucity of oligodendrocytes in the demyelinated
foci. N o picornavirus-like structures or crystalline arrays of virus could be identified. In contrast, none of
the normal Balbk mice became clinically ill or died.
Several of the 16 mice killed 3 weeks to 2 months
following inoculation had occasional scattered inflam-
Brief Communication: Roos and Wollmann: DA Virus Demyelination
495
matory foci, and only 2 had rare naked demyelinated
axons.
Discussion
DA strain of TMEV, a picornavirus, produces a slowly
progressive demyelination. The relative importance of
a viral oligodendrocyte lytic infection and of immunopathological mechanisms in producing the demyelination is unclear. On one hand, Lipton and Dal
Canto have stressed the importance of immunemediated demyelination for the following reasons: at
the time of demyelination, the immune response is
496 Annals of Neurology Vol 15 N o 5 May 1984
Fig 2. Higher magn$cation of the demyelinatedfocusin Figure
1, demonstrating demyelinated axons (arrowheads) and degenerating myelin sheaths (arrows). (Tohidine blue-stained
semithin section of Epon-embe&d tissue; X 2,750 befare 5% reduction.)
brisk, whereas viral titers are low 171;viral antigen in
demyelinated areas is present primarily in macrophages, not in oligodendrocytes [l, 31; demyelination is spatially and temporally associated with
mononuclear infiltrates [2]; by electron microscopy
macrophages can be seen to strip myelin lamellae, as
Fig 3. Degenerating myelin sheath suwounding intact axon in
spinal cord of Nude mouse 2 monthsfillowing inoculation.
( x 18,000 before 5 % reduction.)
has been described in experimental allergic encephalomyelitis [ 2J; and immunosuppression with cyclophosphamide or antithymocyte serum prevents demyelination {S]. O n the other hand, studies have suggested
that DA virus oligodendrocyte infection leads to demyelination: demyelination is present in immunosuppressed DA-infected animals, although reduced in se-
verity, and this demyelination can progress [101; virus
and virus mutants can be isolated from demyelinated
animals {7,11J; and viral antigen is present in oligodendrocytes of chronically demyelinated animals C91.
The present study demonstrates that DA virus produces a chronic, primary demyelination in Nude mice.
The lack of T lymphocyte function and of B-dependent
T cell function in the Nude mice, as well as the absence
of macrophage stripping of myelin lamellae in lesions,
suggests that a direct DA viral oligodendrocyte lytic
infection produces the demyelination.
The finding of DA virus demyelination in Nude
Brief Communication: Roos and Wollmann: DA Virus Demyelination
497
mice is not unexpected, because our previous immunosuppression studies suggested that demyelination
could occur in immunosuppressed animals { 101. The
previous studies, however, demonstrated a decrease in
severity of demyelination in immunosuppressed animals, indicating that immune factors are also important
in the demyelination seen in non-Nude mice {lo]. It is
probable that DA virus demyelinating disease, like
other viral-induced experimental demyelinating diseases, involves multiple mechanisms, perhaps changing
with time and host genotype. For example, the early
necrotic lesions of canine distemper virus encephalo-
498 Annals of Neurology Vol 15 No 5 May 1984
Fig 4. Several demyelinated axons are visible adjacent to an axon
suwounded by an intact myelin sheath in spinal cord of Nude
moue 2 months after inoculation. ( x 18,000 before j% reduction.)
myelitis are not thought to be immune mediated, although the pathogenesis of a late subacute demyelinating encephalomyelitis may involve the immune system
{41. Similarly,JHM strain of mouse hepatitis virus produces acute demyelination in Swiss mice by means of
oligodendroglial cell lysis { 131, although a subacute demyelinating encephalomyelitis following inoculation of
a JHM temperature-sensitive mutant into rats may be
secondary to autoimmune factors E12). In an analogous
fashion, the demyelination of multiple sclerosis may
involve different mechanisms that are active at different times.
Supported by Grant MS:RG1512-A-1 from the National Multiple
Sclerosis Society and by the Kroc Foundation.
The secretarial help of Zayda Stewart is gratefully acknowledged.
The authors also acknowledge the excellent technical assistance of
Joyce Craig.
References
1. Dal Canto MC: Uncoupled relationship between demyelination
and primary infection of myelinating cells in Theiler’s virus encephalomyelitis. Infect Immun 35:1133-1138, 1982
2. Dal Canto MC, Lipton H L Primary demyelination in Theiler’s
virus infection: an ultrastructural study. Lab Invest 33:626-637,
1975
3. Dal Canto MC, Lipton HL: Ultrastructural localization of virus
in acute and chronic demyelinating Theiler’s virus infection. Am
J Pathol 106:20-29, 1982
4. Dal Canto MC, Rabinowitz SG: Experimental models of virusinduced demyelination of the central nervous system. Ann
Neurol 11:109-127, 1982
5. Latov N, Braun PE, Gross RB, et al: Plasma cell dyscrasia and
peripheral neuropathy: identification of the myelin antigens that
react with human paraproteins. Proc Natl Acad Sci USA
78:7139-7142, 1981
6. Lehrich JR, Arnason BGW, Hochberg FH: Demyelinative myelopathy in mice induced by DA virus. J Neurol Sci 29:149160, 1976
7. Lpton HL Theiler’s virus infection in mice: an unusual biphasic
disease process leading to demyelination. Infect Immun
11:1147-1155, 1975
8. Lipton HL, Dal Canto MC: Theiler’s virus-induced demyelination: prevention by immunosuppression. Science 192:62-64,
1976
9. Rodriguez M, Leibowitz JL, Lampert PW: Persistent infection of
oiigodendrocytes in Theiler’s virus-induced encephalomyelitis.
Ann Neurol 13:426-433, 1983
10. Roos RP, Firestone S, Wollmann R, et al: The effect of shonterm and chronic immunosuppression on Theiler’s virus demyelination. J Neuroimmunol 2:223-234, 1982
11. Roos RP, Richards OC, Ehrenfeld E: Analysis of Theiler’s virus
isolates from persistently infected mouse nervous tissue. J Gen
Virol 64:701-706, 1983
12. Watanabe R, Wege H, Ter Meulen V: Coronavirus-host relationship in demyelinating encephalomyelitis of rats: analysis of
CMI reactions to myelin and viral antigens (abstract D3-15).
IXth International Congress of Neuropathology (Vienna) 1982
13. Weiner LP: Pathogenesis of demyelination induced by a mouse
hepatitis virus OHM virus). Arch Neurol 28:298-303, 1973
Reversible Visual Evoked
Potential Abnormalities in
Vitamin E Deficiency
John A. Messenheimer, MD, R. S. Greenwood, MD,
M. B. Tennison, MD, J. J. Brickley, PhD,
and C. J. Ball, R EEG T
~~
A patient with cystic fibrosis and cirrhosis developed a
progressive neurological syndrome associated with
ataxia, proximal weakness, and ophthalmoplegia. Profound deficiencies of vitamins A, D, and E were present.
Visual acuity and results of retinal funduscopy were normal. The pattern reversal visual evoked potential was
initially abnormal (P100 latency, 136 and 130 ms from
left and right eyes, respectively) but became normal (less
than 3 standard deviations from mean control PI00 latency) over a two-month period when vitamin E was
administered. This case documents a potentially reversible visual evoked potential abnormality in a visually
asymptomatic patient with vitamin E deficiency.
Messenheimer JA, Greenwood RS, Tennison MB,
Brickley JJ, Ball CJ: Reversible visual evoked
potential abnormalities in vitamin E deficiency.
A n n Neurol 15:499-501, 1984
Vitamin E deficiency has been implicated as a
pathophysiological agent in certain neurological deficits
encountered in patients with abetalipoproteinemia,
biliary atresia, and cystic fibrosis El, 3, 4, 5 , 12, 141.
Affected patients develop a progressive spinocerebellar degeneration characterized by areflexia, decreased
proprioceptive and vibratory sensation, proximal weakness, gaze paresis, and, in severe cases, retinitis pigmentosa [I, 3 , 4, 5 , 12, 141. Neuropathological examinations have revealed retinal degeneration, degeneration of posterior columns, loss of large-caliber
myelinated fibers in peripheral nerve, and formation of
z o n a l spheroids in the gracile and cuneate nuclei C 1, 3,
4, 12, 131. These lesions are similar to those occurring
in animals with experimental vitamin E deficiency [lo,
111. In some cases, treatment with high doses of watersoluble vitamin E has apparently slowed or arrested
progression of the neurological abnormalities [4, 8, 9,
12, 131. We have examined a patient with cystic
fibrosis and cirrhosis who developed a progressive
neurological syndrome suggestive of vitamin E
From the Department of Neurology, University of North Carolina at
Chapel Hill, Chapel Hill, N C 27514.
Received May 24, 1983, and in revised form Sept 22. Accepted for
publication Sept 23, 1983.
Address reprint requests to Dr Messenheimer.
499
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strait, induced, murine, mice, virus, nude, encephalomyelitis, theiler, demyelination
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