Measles virus-induced hippocampal neurodegeneration in the mouse: A novel, subacute model for testing neuroprotective agents

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Neuroscience Letters, 154 (1993) 109-112 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00

NSL 09494

Measles virus-induced hippocampal neurodegeneration in the mouse: a novel, subacute model for testing neuroprotective agents T o m m y Andersson a, R o b e r t Schwarcz b, Arthur L r v e ¢ and Krister Kristensson a aClinical Research Center, Karolinska lnstitutet, Huddinge Hospital Huddinge (Sweden), bMaryland Psychiatric Research Center, University of Maryland School of Medicine. Baltimore, MD 21228 (USA) and CDepartment of Medical Virology, School of Medicine, University of Iceland, Reykjavik (Iceland) (Received 28 October 1992; Revised version received 5 February 1993; Accepted 10 February 1993)

Key words." Dizocilpine (MK-801); Excitotoxicity; Hippocampus; Measles virus; Neurodegeneration; Neuroprotection The hamster neurotropic (HNT) strain of measles virus causes non-inflammatory encephalopathy in Balb/c mice, associated with neurodegeneration in hippocampal CA 1 and CA3 regions. This loss of pyramidal cells can be prevented by twice daily systemic treatment with 1 mg/kg dizocilpine (5-methyl- 10,11-dihydro-5H-dibenzo(a,d)cyclo-hepten-5,10-imine maleate; MK-801) for 7 days. By varying the MK-801 treatment protocol, we now found that drug administration during the last 4 days prior to sacrifice (i.e. days 4-7 post inoculation, p.i.) is essential for neuroprotection. In contrast, MK-801 treatment during the first days (days 0 ~ p.i.) did not prevent the neuronal necrosis. These data suggest that the concentration of an excitotoxic factor in the mouse brain increases after virus inoculation, reaching toxic levels by days 4-5 p.i. This novel 'subacute' mouse model of neurodegeneration therefore constitutes an attractive tool for mechanistic and interventional studies in excitotoxicity research.

By 5 days after an intracerebral inoculation of the hamster neurotropic (HNT) strain of measles virus in Balb/c mice, animals develop a non-inflammatory encephalopathy associated with severe hyperexcitation and sporadic seizure activity [3, 5]. Virus antigen is located in clusters of neurons, mainly in the cerebral cortex, whereas degeneration of neurons occurs in the CA1 and CA3 sectors of the hippocampal pyramidal cell layer [4]. This pattern of neuronal loss resembles that seen after an intrahippocampal application of excitatory amino acids, in particular quinolinate [6]. In a previous study [1], we showed that this neurodegeneration can be prevented by continuous, twice daily treatment with dizocilpine (5-methyl- 10,11-dihydro-5H-dibenzo(a,d)cyclo-hepten-5,10-imine maleate; MK-801), a non-competitive NMDA-receptor antagonist [8]. It was concluded that intracerebral virus application causes neurodegeneration indirectly due to activation of NMDA-receptors. The present study was designed to further examine the mechanism of virus-induced nerve cell loss by varying the MK-801 treatment protocol to include drug-free periods in the early or late stages of the degenerative procCorrespondence: T. Andersson, Clinical Research Center, Huddinge Hospital F41, S-141 86 Huddinge, Sweden.

ess. This work has revealed notable features of the probable sequence of neurodegenerative events which take place during the first 7 days after virus inoculation. The experimental procedures of virus inoculation have been described previously [1]. A total of 63 female Balb/c mice were inoculated during deep ether anesthesia, as judged by absence of the pedal withdrawal reflex, with a brain homogenate containing the HNT strain of measles virus, and subsequently divided at random into eight groups. Four animals were inoculated with brain homogenate containing virus previously inactivated by ultraviolet light. Mice in all groups were injected intraperitoneally with 1.0 mg of MK-801 (RBI, Natick, MA) per kg body weight or saline as follows: Group I: MK-801, day 0-7 p.i., n = 4 Group II: MK-801, day 2-7 p.i., n = 6 Group III: MK-801, day 4-7 p.i., n = 9 Group IV: MK-801, day 6-7 p.i., n = 8 Group V: MK-801, day 0-2 p.i., n --- 9 Group VI: MK-801, day 0-4 p.i., n = 9 Group VII: MK-801, day 0-6 p.i., n = 9 Group VIII: Saline, day 0-7 p.i., n = 8 Group IX: Inactivated virus, saline, day 0-7, n = 4 All animals received either saline or MK-801 at regular 12-h intervals, starting immediately after virus inocu-

I10 lation. The animals were observed daily for clinical signs o f disease. After 7 days, the mice were decapitated, their brains dissected and immersed in 4% p a r a f o r m a t d e h y d e (in 0,2 M S6rensen's phoshate buffer) for 24 h, rinsed and kept in 15% sucrose for at least 24 h. Subsequently coronal cryostat sections ( 2 0 / l m ) , representing 12 15 levels o f the hippocampus, were cut, stained with Cresyl

violet and m o u n t e d in P e r m o u n t (Fischer Scientific, N J, USA). Stereological evaluations were made with a Reichert-Jung Visopan microscope. Six sections, evenly distributed within each hippocampal formation, were used for calculation of the volume density o f the CA1 and CA3 neurons as detailed by Weibel [7]. As described before [1], animals developed virus-in-

Fig. 1. Micrographs of Cresyl violet-stained sections showing the extent of neurodegeneration in the hippocampus of infected control (A,B) and MK-g01-treated (C H) mice at 7 days p.i. B, D, F and H are higher magnifications of the areas indicated by boxes in A, C, E and G, respectively. The illustrations depict typical examples from MK-801-treated groups V1 (C,D), VII (E,F), and lII (G,H). Note the partial protection of pyramidal cells in E and F and the normal appearance of the pyramidal cell layer in G and H. Bar = 100/tin (A, C, E and G) and 50,urn (B, D, F and H).

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duced hyperexcitation or hyperirritation, combine davith sporadic seizures starting on day 5 p.i. No differences in overt clinical symptoms were noted between infected (saline-injected) controls and infected MK-801-treated mice. On day 7 p.i., the animals gradually entered a lethargic state with low responsiveness to external stimuli. The distribution of hippocampal neurodegeneration in the infected control brains was similar to that observed previously (Fig. 1A,B of ref. 1). Thus, bilateral lesions occurred in the CA1 and CA3 areas of the pyramidal band, whereas the CA2 and granule cells were relatively spared. The degeneration was more pronounced in CA1 than CA3, and no inflammatory cells were seen. Stereological calculations (Fig. 2), measuring the relative volume of these areas, revealed that the saline-treated, infected group (VIII) was significantly different from uninfected controls (group IX; P < 0.001). Provided that the inoculum does not contain a component other than the virus which is inactivated by ultraviolet light, this indicates that the lesions are related to virus replication in the brain. Groups IV, V and VI (receiving MK-801 on days 6-7, 0-2 and 0-4, respectively), also showed marked cell loss (Fig. 1C,D) (P < 0.001 vs. uninfected controls). Group VII (receiving MK-801 on days 0-6) displayed

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Fig. 2. Stereologicalmeasurementsof hippocampal neurodegeneration in the pyramidal cell layer. The volume densities of the CA1 and CA3 regions, i.e. their relativevolume as compared to the whole hippocampal volume,are presentedas a percentageof that of uninfectedcontrols (group IX, n -- 4). Groups I, II and III, receivingMK-801 on days 0-7, 2-7 and 4~7, respectively,displayed no significant neuronal loss. In contrast, groups IV, V and VI, receivingthe drug on days 6-7, 0-2 and 04, respectively,and the saline-treated group (VIII), showed significant neurodestruction.Group VII, receivingMK-801 in days 0-6, presented less prominent but still significantnerve cell loss. Data are presented as the mean _+S.E.M. ***P < 0.001; *P < 0.05, as compared to controls (Student's t-test), ns, not significant.

less conspicuous h u t still significant (P < 0.05) cell loss (Fig. 1E,F). In contrast, groups I, II and III (receiving MK-801 on days 0-7, 2-7 and 4-7, respectively) had no significant CA1 and CA3 degeneration (Fig. 1G,H) as compared to uninfected controls. In this study, we verified that repeated intraperitoneal administration of MK-801 protects against measles virus-induced hippocampal neurodegeneration. Furthermore, our data indicate that drug treatment during days 4~7 p.i. is crucial for neuroprotection in this mouse model. Even withdrawal of MK-801 for only the last day before sacrifice led to a significant lesion of CA1 and CA3 neurons. On the other hand, MK-801 administration during the first 4 days is clearly not essential for neuroprotection in our paradigm. It therefore appears that the cellular and molecular events resulting in excessive N M D A receptor activation do not become sufficiently prominent or irreversible prior to day 5 p.i. In this context, it is noteworthy that hypertrophied astrocytes are seen as early as 3 days p.i., that the astrocytic response to the virus increases in intensity in the course of the following days, and that neuroprotective doses of MK-801 do not affect the virus-induced astrocytic reaction [1]. Moreover, reactive astrocytes contain the enzyme responsible for the synthesis of the endogenous excitotoxin quinolinic acid [2]. Taken together with the present results, it therefore appears plausible that the concentration of an astrocyte-derived factor (such as quinolinic acid) increases in the course of the post-inoculation period, reaching lethally toxic levels by day 5 p.i. As compared to other experimental situations in which hippocampal pyramidal cell degenerate, for example ischemia, hypoglycemia, limbic seizures and intrahippocampal injection of excitotoxic amino acids, the model described here offers several advantages. First the animals are awake and unsedated during the experiment and can therefore be easily studied and manipulated. Second, the clinical signs and morphological lesions have a high degree of reproducibility, and the inductive agent, i.e. the virus, is easily administered. Most importantly, the H N T virus model is 'subacute', i.e. the animals develop neuropathological changes and associated clinical characteristics relatively slowly over a period of several days. This provides a temporally significant window of opportunity both for mechanistic studies of the neurodegenerative process and for intervention with potential neuroprotective drugs. Thus, while cell death in this model may also be critically influenced by virus-derived factors other than the essential MK-801-sensitive component, it seems advisable to add the measles virus-infected mouse to the experimental armamentarium for excitotoxicological research.

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1 Andersson, Y., Schultzberg, M., Schwarcz, R., L6ve, A., Wickman, C. and Kristensson, K., NMDA-receptor antagonist prevents measles virus-induced neurodegeneration, Eur. J. Neurosci., 3 (1991) 6671. 2 Eastman, C.L., Du, F., Andersson, T., L6ve, A. and Schwarcz, R., Increased 3-hydroxyanthranilic oxygenase immunoreactivity in measles-virus infected mice, Soc. Neurosci. Abstr., 18 (1992) 667.3. 3 Griffin, D.E., Mullinix, J., Narayan, O. and Johnson, R.T., Age dependence of viral expression: comparative pathogenesis of two rodent-adapted strains of measles virus in mice, Infect. Immunol.. 9 (1974) 690 695.

4 L6ve, A., Norrby, E. and Kristensson, K., Measles encephalitis in rodents: defective expression of viral proteins, J. Neuropathol. Exp. Neurol., 45 (1986) 258-267. 5 Rammohan, K.W., McFarlin, D.E. and McFarland, H.F., Chronic measles encephalitis in mice, J. Infect. Dis., 142 (1980) 608-613. 6 Schwarcz, R., Brush, G.S., Foster, A.C. and French, E.D., Seizure activity and lesions after intrahippocampal quinolinic acid injection, Exp. Neurol., 84 (1984) 1 17. 7 Weibel, E.R., Stereological methods, Vol. 1, Practical Methods tbr Biological Morphometry, Academic Press, London, 1979, 415 pp. 8 Wong, E.H., Kemp, J.A., Priestley, T., Knight, A.R., Woodruff, G.N. and Iversen, L.L., The anticonvulsant MK-801 is a potent Nmethyl-D-aspartate antagonist, Proc. Natl. Acad. Sci. USA, 83 (1986) 7104-7108.