Acute hemorrhagic demyelination in a murine model of multiple sclerosis
© Pirko et al; licensee BioMed Central Ltd. 2008
Received: 07 May 2008
Accepted: 07 July 2008
Published: 07 July 2008
Acute hemorrhagic leukoencephalomyelitis (AHLE) is a rare neurological condition characterized by the development of acute hemorrhagic demyelination and high mortality. The pathomechanism of AHLE, as well as potential therapeutic approaches, have remained elusive due to the lack of suitable animal models. We report the first murine model of AHLE using a variation of the Theiler's Murine Encephalitis Virus (TMEV) MS model. During acute TMEV infection, C57BL/6 mice do not normally undergo demyelination. However, when 7 day TMEV infected C57BL/6 mice are intravenously administered the immunodominant CD8 T cell peptide, VP2121–130, animals develop characteristics of human AHLE based on pathologic, MRI and clinical features including microhemorrhages, increased blood-brain barrier permeability, and demyelination. The animals also develop severe disability as assessed using the rotarod assay. This study demonstrates the development of hemorrhagic demyelination in TMEV infected C57BL/6 mice within 24 hours of inducing this condition through intravenous administration of CD8 T cell restricted peptide. This study is also the first demonstration of rapid demyelination in a TMEV resistant non-demyelinating strain without transgenic alterations or pharmacologically induced immunosuppression.
The acute monophasic demyelinating disorders, including acute disseminated encephalomyelitis (ADEM) and acute hemorrhagic leukoencephalitis (AHLE) usually present 1–3 weeks after infections or vaccination, but have also been observed without preceding illness [1, 2]. In cases of ADEM, the prognosis is favorable, with 60–80% of cases experiencing complete recovery [3, 4]. However, AHLE is associated with rapidly deteriorating focal and diffuse neurological symptoms leading to death within 2–14 days [5–8]. The few patients that survive AHLE usually have significant residual neurological symptoms . Its high mortality and poor response to therapy necessitate the development of animal models of AHLE to understand the mechanism of its pathology.
During acute TMEV infection of the H-2b haplotype, 50–70% of central nervous system (CNS) infiltrating CD8+ T cells have T cell receptor specificity towards an immunodominant viral peptide VP2121–130 presented in the context of the Db class I molecule . We have previously reported that a rapidly fatal hemorrhagic CNS disease develops in the C57BL/6 strain when the immunodominant VP2121–130 peptide is intravenously administered 7 days post TMEV infection . In these studies, we confirmed by northern blot analysis that TMEV RNA in the CNS was not increased in animals administered VP2121–130 peptide, demonstrating that this fatal condition was not due to increased viral load . The C57BL/6 strain is considered non-demyelinating, as these mice do not develop chronic viral persistence and demyelination . We now report that the fatal hemorrhagic CNS disease in these mice is associated with demyelination. Our findings highlight two very important concepts: 1) a classically non-demyelinating strain can develop fulminant hemorrhagic demyelination by intravenous administration of an immunodominant peptide recognized by CD8 T cells; and 2) this hyperacute model of hemorrhagic demyelination is the first TMEV-induced murine model of AHLE.
All experiments were approved by the Institutional Animal Care and Use Committee of the University of Cincinnati. All adequate measures were taken to minimize pain or discomfort, and experiments were conducted in accordance with international standards on animal welfare as well as being compliant with local and national regulations. TMEV infection was induced via intracranial injection of 2 × 106 PFUs of TMEV . Induction of the hemorrhagic demyelinating condition required IV injection of 0.1 mg VP2121–130 peptide 7 days after infection. Db binding Human papilloma virus E7 peptide was used as negative control .
(2) To determine the presence and extent of demyelination, mice were perfused via intracardiac puncture with 50 mL Trump's fixative solution. Brains were removed and post-fixed for an additional 24 hours in Trump's fixative. Coronal blocks of brain tissue were osmicated and embedded in glycol methacrylate . Two-μm-thick sections were stained with a modified paraphenolyene diamine stain to detect demyelination.
We report that immunodominant peptide injection in TMEV infected C57BL/6 mice causes significant blood brain barrier (BBB) permeability and CNS damage, resulting not only in inflammatory infiltrates, microhemorrhages, and tissue damage, but also demyelination. To our knowledge, this is the first example of inducing demyelination in a non-demyelinating mouse strain in the TMEV model without the use of immunosuppressants or transgenic technology . Our previously published results also demonstrate that CD8+ T cells specific for the immunodominant Db: VP2 121–130 epitope play a key role in mediating the observed pathology .
Demyelination is rapid in this murine model, occurring within 24 hours of intravenous injection of VP2 peptide. Also of interest, demyelination, as a result of TMEV infection, is normally not observed in the C57BL/6 strain. Susceptibility to demyelination in the TMEV model is largely dependent on the major histocompatibility class I genotype of the mouse . However, other quantitative trait loci (QTL) pertaining to susceptibility to persistent virus infection and chronic demyelination in the spinal cord have also been defined . C57BL/6 mice, being of the resistant H-2b haplotype, mount a very strong antiviral response, mediated by epitope specific CD8+ T-cells recognizing the VP2121–130 viral capsid fragment . This results in clearance of the virus, and survival of the animal without neurological deficits. As demonstrated by this study, a demyelinating syndrome can be rapidly induced by a simple in vivo injection of the immunodominant peptide VP2 which is recognized by the majority of brain infiltrating CD8 T cells .
In the human condition, ADEM is characterized by multifocal perivascular demyelination in an asymmetric fashion, mostly in the white matter. However, gray matter involvement of the basal ganglia, thalamus and brainstem has also been reported. In AHLE, necrotizing immune infiltration, perivascular demyelination, ball and ring hemorrhages, prominent infiltrates with lymphocytes, macrophages and neutrophils have been reported . Radiologically AHLE is also more severe than ADEM, with mass effect, edema, and punctuate hemorrhages being present adjacent to usually asymmetric T2 hyperintense lesions. The histological and MRI findings put forth in this study clearly present with these traits, demonstrating that in vivo activation of CNS infiltrating CD8 T cells can serve as a novel model of AHLE. Such studies may ultimately lead to the development of more effective and focused therapies instead of non-specific and partially effective use of steroids , cyclophosphamide or plasma exchange . Future experiments directed at putative mechanisms of BBB disruption and demyelination are already underway in our labs.
We would like to thank R. Scott Dunn and Ron Pratt for their technical assistance with MRI acquisition. We would also like to thank Hollis Miller for assistance in compliance issues and maintenance of our vertebrate studies. We would like to thank Mabel Pierce for the histologic preparations. This work was funded through a grant awarded by The Neuroscience Institute (TNI) of Cincinnati and the National Institutes of Health RO1 #NS058698.
- Kesselring J, Miller DH, Robb SA, Kendall BE, Moseley IF, Kingsley D, du Boulay EP, McDonald WI: Acute disseminated encephalomyelitis. MRI findings and the distinction from multiple sclerosis. Brain. 1990, 113 (Pt 2): 291-302. 10.1093/brain/113.2.291.View ArticlePubMedGoogle Scholar
- Gibbs WN, Kreidie MA, Kim RC, Hasso AN: Acute hemorrhagic leukoencephalitis: neuroimaging features and neuropathologic diagnosis. J Comput Assist Tomogr. 2005, 29: 689-693. 10.1097/01.rct.0000173843.82364.db.View ArticlePubMedGoogle Scholar
- Tenembaum S, Chamoles N, Fejerman N: Acute disseminated encephalomyelitis: a long-term follow-up study of 84 pediatric patients. Neurology. 2002, 59: 1224-1231.View ArticlePubMedGoogle Scholar
- Dale RC: Acute disseminated encephalomyelitis. Semin Pediatr Infect Dis. 2003, 14: 90-95. 10.1053/spid.2003.127225.View ArticlePubMedGoogle Scholar
- Hart MN, Earle KM: Haemorrhagic and perivenous encephalitis: a clinical-pathological review of 38 cases. J Neurol Neurosurg Psychiatry. 1975, 38: 585-591.PubMed CentralView ArticlePubMedGoogle Scholar
- Suchowersky O, Sweeney VP, Berry K, Bratty PJ: Acute hemorrhagic leukoencephalopathy. A clinical, pathological, and radiological correlation. Can J Neurol Sci. 1983, 10: 63-67.PubMedGoogle Scholar
- Geerts Y, Dehaene I, Lammens M: Acute hemorrhagic leukoencephalitis. Acta Neurol Belg. 1991, 91: 201-211.PubMedGoogle Scholar
- Posey K, Alpert JN, Langford LA, Yeakley JW: Acute hemorrhagic leukoencephalitis: a cause of acute brainstem dysfunction. South Med J. 1994, 87: 851-854. 10.1097/00007611-199408000-00023.View ArticlePubMedGoogle Scholar
- Rosman NP, Gottlieb SM, Bernstein CA: Acute hemorrhagic leukoencephalitis: recovery and reversal of magnetic resonance imaging findings in a child. J Child Neurol. 1997, 12: 448-454.View ArticlePubMedGoogle Scholar
- Johnson AJ, Njenga MK, Hansen MJ, Kuhns ST, Chen L, Rodriguez M, Pease LR: Prevalent class I-restricted T-cell response to the Theiler's virus epitope Db:VP2121-130 in the absence of endogenous CD4 help, tumor necrosis factor alpha, gamma interferon, perforin, or costimulation through CD28. J Virol. 1999, 73: 3702-3708.PubMed CentralPubMedGoogle Scholar
- Johnson AJ, Mendez-Fernandez Y, Moyer AM, Sloma CR, Pirko I, Block MS, Rodriguez M, Pease LR: Antigen-specific CD8+ T cells mediate a peptide-induced fatal syndrome. J Immunol. 2005, 174: 6854-6862.View ArticlePubMedGoogle Scholar
- Brahic M, Bureau JF, Michiels T: The genetics of the persistent infection and demyelinating disease caused by Theiler's virus. Annu Rev Microbiol. 2005, 59: 279-298. 10.1146/annurev.micro.59.030804.121242.View ArticlePubMedGoogle Scholar
- Pirko I, Fricke ST, Johnson AJ, Rodriguez M, Macura SI: Magnetic resonance imaging, microscopy, and spectroscopy of the central nervous system in experimental animals. NeuroRx. 2005, 2: 250-264. 10.1602/neurorx.2.2.250.PubMed CentralView ArticlePubMedGoogle Scholar
- Robb RA: 3-D visualization in biomedical applications. Annu Rev Biomed Eng. 1999, 1: 377-399. 10.1146/annurev.bioeng.1.1.377.View ArticlePubMedGoogle Scholar
- Marti M, Mela F, Veronesi C, Guerrini R, Salvadori S, Federici M, Mercuri NB, Rizzi A, Franchi G, Beani L, et al: Blockade of nociceptin/orphanin FQ receptor signaling in rat substantia nigra pars reticulata stimulates nigrostriatal dopaminergic transmission and motor behavior. J Neurosci. 2004, 24: 6659-6666. 10.1523/JNEUROSCI.0987-04.2004.View ArticlePubMedGoogle Scholar
- Onyszchuk G, Al-Hafez B, He YY, Bilgen M, Berman NE, Brooks WM: A mouse model of sensorimotor controlled cortical impact: characterization using longitudinal magnetic resonance imaging, behavioral assessments and histology. J Neurosci Methods. 2007, 160: 187-196. 10.1016/j.jneumeth.2006.09.007.PubMed CentralView ArticlePubMedGoogle Scholar
- Rodriguez M: Immunoglobulins stimulate central nervous system remyelination: electron microscopic and morphometric analysis of proliferating cells. Lab Invest. 1991, 64: 358-370.PubMedGoogle Scholar
- Pirko I, Johnson A, Ciric B, Gamez J, Macura SI, Pease LR, Rodriguez M: In vivo magnetic resonance imaging of immune cells in the central nervous system with superparamagnetic antibodies. Faseb J. 2004, 18: 179-182.PubMedGoogle Scholar
- Drescher KM, Pease LR, Rodriguez M: Antiviral immune responses modulate the nature of central nervous system (CNS) disease in a murine model of multiple sclerosis. Immunol Rev. 1997, 159: 177-193. 10.1111/j.1600-065X.1997.tb01015.x.View ArticlePubMedGoogle Scholar
- Dethlefs S, Escriou N, Brahic M, Werf van der S, Larsson-Sciard EL: Theiler's virus and Mengo virus induce cross-reactive cytotoxic T lymphocytes restricted to the same immunodominant VP2 epitope in C57BL/6 mice. J Virol. 1997, 71: 5361-5365.PubMed CentralPubMedGoogle Scholar
- Huang CC, Chu NS, Chen TJ, Shaw CM: Acute haemorrhagic leucoencephalitis with a prolonged clinical course. J Neurol Neurosurg Psychiatry. 1988, 51: 870-874.PubMed CentralView ArticlePubMedGoogle Scholar
- Seales D, Greer M: Acute hemorrhagic leukoencephalitis. A successful recovery. Arch Neurol. 1991, 48: 1086-1088.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.