A Murine Model of Retroviral Encephalopathy

Yoshitatsu Sei1, Anthony S. Basile2

1 Uniformed Services University of the Health Sciences, Bethesda, Maryland, 2 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 9.8
DOI:  10.1002/0471142301.ns0908s17
Online Posting Date:  February, 2002
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Abstract

This unit delineates the steps for production of a murine model of retroviral encephalopathy. The LP‐BM5 infected mouse develops a chronic inflammation of the brain secondary to profound immune deficiency. The model is robust, develops rapidly and does not require the use of human pathogens. In addition, the behavioral and neurochemical characteristics of this model is reviewed.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of a High‐Titer LP‐BM5 Inoculate and Infection of C57BL/6 Mice
  • Support Protocol 1: Preparation of LP‐BM5‐Infected and Uninfected SC‐1 Cell Lines
  • Basic Protocol 2: Monitoring of LP‐BM5 Infection and Disease Progression in Mice by Spleen Size
  • Basic Protocol 3: Monitoring of LP‐BM5 Infection and Disease Progression in Mice by T and B Cell Populations
  • Basic Protocol 4: Monitoring Virus Burden of LP‐BM5‐Infected Mice for def‐gag by PCR
  • Basic Protocol 5: Behavioral Monitoring of Encephalopathy
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of a High‐Titer LP‐BM5 Inoculate and Infection of C57BL/6 Mice

  Materials
  • Uninfected and LP‐BM5‐infected SC‐1 cultures (see protocol 2)
  • 0.25% (w/v) trypsin/1 mM EDTA, 37°C (Life Technologies)
  • Complete Dulbecco's modified Eagle medium (DMEM; Quality Biological or appendix 2A)
  • C57BL/6 mice, 4 to 6 weeks postnatal age (Jackson Laboratories or Charles River Laboratories)
  • 37°C, 5% CO 2 incubator
  • 50‐ml conical tubes
  • 150‐cm2 flasks
  • Cell scraper (Corning)
  • 1.5‐ml cryotubes (Nalgene/Nunc)
  • 37°C water bath
  • 1‐ and 3‐ml syringes with 25‐G and 22‐G needles, respectively
  • Additional reagents and equipment for determining cell number and viability with a hemacytometer and trypan blue staining ( appendix 3B) and monitoring MAIDS progression (see protocol 3Basic Protocols 2 to protocol 65)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Preparation of LP‐BM5‐Infected and Uninfected SC‐1 Cell Lines

  • Mouse embryonic fibroblast‐like cells (SC‐1 cells; ATCC #CRL‐1404)
  • SC‐1 cells chronically infected with MuLV‐BM5 (SC‐1/MuLV LP‐BM5; NIH AIDS Reference Reagent Program #1215)
  • Hanks balanced salt solution (HBSS; Quality Biological or appendix 2A)

Basic Protocol 2: Monitoring of LP‐BM5 Infection and Disease Progression in Mice by Spleen Size

  Materials
  • LP‐BM5‐infected C57BL/6 mice (see protocol 1)
  • 5 mg/ml sodium pentobarbital
  • 70% ethanol
  • Rodent guillotine
  • Toothed forceps and surgical scissors, sterile

Basic Protocol 3: Monitoring of LP‐BM5 Infection and Disease Progression in Mice by T and B Cell Populations

  Materials
  • LP‐BM5‐infected C57BL/6 mice (see protocol 1)
  • 5 mg/ml sodium pentobarbital
  • 70% ethanol
  • DMEM (Quality Biological or appendix 2A)
  • ACK lysis buffer (Quality Biological)
  • Hanks' balanced salt solution (HBSS; Sigma or appendix 2A)
  • Fluorescent‐tagged monoclonal antibody (e.g., FITC‐Ly6D, FITC‐CD4, FITC‐CD8a, FITC‐Mac3, PE‐CD25, PE‐I‐Ab)
  • 0.4% paraformaldehyde in PBS ( appendix 2A), 0° to 4°C
  • Surgical scissors and toothed forceps, sterile
  • Small plastic petri dish or 6‐well plate (Costar)
  • 3‐ml plastic syringe plunger
  • 15‐ml centrifuge tube, sterile
  • 12 × 75–mm conical tubes without caps (Becton Dickinson)
  • Flow cytometer (Becton Dickinson)
  • Additional reagents and equipment for determining cell number and viability with a hemacytometer and trypan blue staining ( appendix 3B)

Basic Protocol 4: Monitoring Virus Burden of LP‐BM5‐Infected Mice for def‐gag by PCR

  Materials
  • Uninfected and LP‐BM5‐infected C57BL/6 mice (see protocol 1)
  • Phosphate buffered saline (PBS; Digene or appendix 2A)
  • Total RNA isolation kit (e.g., Promega SV Total RNA Isolation System; optional)
  • cDNA synthesis kit (e.g., Pharmacia First‐Strand)
  • 15 pmol/µl primer solutions for defgag:
  • defgag sense sequence: CCT TTT CCT TTA TCG ACA CT
  • defgag antisense sequence: ACC AGG GGG GGA ATA CCT CG
  • 15 pmol/µl primer solutions for β‐actin internal standard (Clonetech)
  •  β‐actin sense sequence: GTG GGC CGC TCT AGG CAC CAA
  •  β‐actin antisense sequence: CTC TTT GAT GTC ACG CAC GAT TTC
  • 10 mM dNTP mixture (Perkin Elmer)
  • 10× PCR buffer (Perkin Elmer)
  • 25 mM MgCl 2
  • 5 U/µl Taq DNA polymerase (Perkin Elmer)
  • Nuclease free‐water (Digene)
  • Small plastic petri dish or 6‐well plate (Costar)
  • MicroAmp reaction tubes (Perkin Elmer)
  • PCR thermal cycler (Perkin‐Elmer)
  • Camera
  • Image analyzer (e.g., MetaMorph, Universal Imaging)
  • Additional reagents and solutions for the acid guanidinium thiocyanate/phenol/chloroform method of isolating total RNA (Chomczynski and Sacchi, ; optional) and agarose gel electrophoresis ( appendix 1N)
NOTE: Use Nuclease free‐pipet tips (i.e., sterile with aerosol‐barrier; Rainin Instruments) for all pipetting steps.

Basic Protocol 5: Behavioral Monitoring of Encephalopathy

  Materials
  • Dry milk powder
  • Uninfected and LP‐BM5‐infected C57BL/6 mice (see protocol 1)
  • Cylindrical black Plexiglas tank with a diameter of 1 m and walls 25 cm high
  • 14‐cm high clear Plexiglas pedestal topped by a 7‐cm diameter platform covered with surgical tape
  • Movement tracking and analysis hardware and software (e.g., Smart/Poly‐track; San Diego Instruments)
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Figures

Videos

Literature Cited

Literature Cited
   Aziz, D.C., Hanna, Z., and Jolicoeur, P. 1989. Severe immunodeficiency disease induced by a defective murine leukemia virus. Nature 338:505‐508.
   Buller, R.M.L., Yetter, R.A., Frederickson, T.N., and Morse, H.C. III. 1987. Abrogation of resistance to severe mouse‐pox in C57BL/6 mice infected with LP‐BM5 murine leukemia viruses. J. Virol. 61:383‐387.
   Choi, D.W. 1992. Excitotoxic cell death. J. Neurobiol. 23:1261‐1276.
   Chomczynski, P. and Sacchi, N. 1987. Single‐step method of RNA isolation by acid guanidinium thiocyanate‐phenol‐chloroform extraction. Anal. Biochem. 162:156‐159.
   Clark, G.D., Happel, L.T., Zorumski, C.F., and Bazan, N.G. 1992. Enhancement of hippocampal excitatory synaptic transmission by platelet‐activating factor. Neuron 9:1211‐1216.
   Cloyed, M.W., Hartely, J.W., and Rowe, W.P. 1981. Genetic study of lymphoma induction by AKR mink cell focus inducing virus in ARK × NFS crosses. J. Exp. Med. 154:450.
   English, J.A., Hemphill, K.A., and Paul, I.A. 1998. LP‐BM5 infection impairs acquisition, but not performance, of active avoidance responding in C57BL/6 mice. F.A.S.E.B. J. 12:175‐179.
   Espey, M.G. and Basile, A.S. 1999. Glutamatergic hyperactivation of the hypothalamic‐pituitary‐adrenal axis in LP‐BM5 infected mice augments immunodeficiency. J. Immunol. 162:4998‐5002.
   Espey, M.G., Kustova, Y., Sei, Y., and Basile, A.S. 1998a. Extracellular glutamate levels are elevated in the brains of LP‐BM5 infected mice: Relevance to retrovirus associated encephalopathies. J. Neurochem. 71:2079‐2087.
   Espey, M.G., Kustova, Y., Sei, Y., and Basile, A.S. 1998b. The LP‐BM5 murine leukemia virus: Impact on the immune and central nervous systems. In Neuroimmunodegeneration (P.K.Y. Wong and W. Lynn, eds.) pp. 95‐114. Springer Verlag, Berlin.
   Grant, S.G.N., O'Dell, T.J., Karl, K.A., Stein, P.L., Soriano, P., and Kandel, E.R. 1992. Impaired long‐term potentiation, spatial learning, and hippocampal development in fyn mutant mice. Science 258:1903‐1910.
   Ha, J.‐H., Sei, Y., and Basile, A.S. 1995. Striatal met‐enkephalin and substance P levels are decreased in mice infected with the LP‐BM5 murine leukemia virus. J. Neurochem. 64:1896‐1898.
   Haas, M. and Meshorer, A. 1979. Reticulum cell neoplasms induced in C57BL/6 mice by cultured virus grown in stromal hematopoietic cell lines. J. Natl. Cancer Inst. 63:427‐439.
   Hanna, Z., Kay, D.G., Rebai, N., Guimond, A., Jothy, S., and Jolicoeur, P. 1998. Nef harbors a major determinant of pathogenicity for an AIDS‐like disease induced by HIV‐1 in transgenic mice. Cell 95:163‐175.
   Hartley, J.W., Frederickson, T.N., Yetter, R.A., Makino, M., and Morse III, H.C. 1989. Retrovirus‐induced murine acquired immunodeficiency syndrome: Natural history of infection and differing susceptibility of inbred mouse strains. J. Virol. 63:1223‐1231.
   Iida, R., Yamada, K., Mamiya, T., Saito, K., Seishima, M., and Nabeshima, T. 1999. Characterization of learning and memory deficits in C57BL/6 mice infected with LP‐BM5, a murine model of AIDS. J. Neuroimmunol. 95:65‐72.
   Kustova, Y., Sei, Y., Goping, G., and Basile, A.S. 1997a. Gliosis in the LP‐BM5 murine leukemia virus‐infected mouse: An animal model of retrovirus‐induced dementia. Brain Res. 742:271‐282.
   Kustova, Y., Espey, M.G., Sei, Y., and Basile, A.S. 1997b. Regional decreases in AMPA receptor density in mice infected with the LP‐BM5 murine leukemia virus. NeuroReport 8:1243‐1247.
   Kustova, Y., Ha, J‐H., Espey, M., Sei, Y., and Basile, A.S. 1998a. The pattern of neurotransmitter alterations in LP‐BM5 infected mice are consistent with glutamatergic hyperactivation. Brain Res. 793:119‐126.
   Kustova, Y., Espey, M.G., Sung, E.‐G., Morse, D., Sei, Y., and Basile, A.S. 1998b. Evidence of neuronal degeneration in C57BL/6 mice infected with the LP‐BM5 leukemia retrovirus mixture. Mol. Chem. Neuropathol. 35:39‐59.
   Kustova, Y., Grinberg, A., and Basile, A.S. 1999. Increased blood‐brain barrier permeability in LP‐BM5 infected mice is mediated by neuroexcitatory mechanisms. Brain Res. 839:153‐163.
   Kustova, Y., Sei, Y., McCarty, T., Espey, M.G., Ming, R., Morse III, H.C., and Basile, A.S. 2000. Accelerated development of neurocognitive deficits in LP‐BM5 infected mice with target deletions of the IFN‐g gene. J. Neuroimmunol. 108:112‐121.
   Lee, B., English, J.A., and Paul, I.A. 2000. LP‐BM5 infection impairs spatial working memory in C57BL/6 mice in the Morris water maze. Brain Res. 856:129‐134.
   Li, Y., Kustova, Y., Sei, Y., and Basile, A.S. 1997. Regional changes in constitutive, but not inducible NOS expression in the brains of mice infected with the LP‐BM5 Leukemia virus. Brain Res. 752:107‐116.
   McArthur, J.C., Selena, O.A., Glass, J.D., Hoover, D.R., and Bacellar, H. 1994. HIV dementia. Incidence and risk factors. Res. Publ. Assoc. Res. Nerv. Ment. Dis. 72:251‐272.
   Morse III, H.C., Chattopadhyay, S.K., Makino, M., Fredrickson, T.N., Hugin, A.W., and Hartley, J.W. 1992. Retrovirus‐induced immunodeficiency in the mouse: MAIDS as a model for AIDS. AIDS 6:607‐621.
   Nishida, K., Markey, S.P., Kustova, Y., Morse III, H.C., Skolnick, P., Basile, A.S., and Sei, Y. 1996. Increased brain levels of platelet‐activating factor in a murine acquired immune deficiency syndrome are NMDA receptor‐mediated. J. Neurochem. 66:433‐435.
   Patneau, D.K. and Mayer, M.L. 1990. Structure‐activity relationships for amino acid transmitter candidates acting at N‐Methyl‐D‐aspartate and quisqualate receptors. J. Neurosci. 10:2385‐2399.
   Piani, D. and Fontana, A. 1994. Involvement of the cystine transport system Xc− in the macrophage induced glutamate‐dependent cytotoxicity to neurons. J. Immunol. 152:3578‐3585.
   Rowe, W.P., Pugh, W.E., and Hartley, J.W. 1970. Plaque assay techniques for murine leukemia viruses. Virology 42:1136‐1139.
   Sei, Y., Arora, P.K., Skolnick, P., and Paul, I.A. 1992a. Spatial learning impairment in a murine model of AIDS. F.A.S.E.B. J. 6:3008‐3013.
   Sei, Y., Makino, M., Vitkovic, L., Chattopadhyay, S.K., Hartley, J.W., and Arora, P.K. 1992b. Central nervous system infection in murine retrovirus‐induced immunodeficiency syndrome. J. Neuroimmunol. 37:131‐140.
   Sei, Y., Paul, I.A., Saito, K., Layer, R., Hartley, J.W., Morse III, H.C., Skolnick, P., and Heyes, M.P. 1996a. Quinolinic acid levels in a murine retrovirus‐induced immunodeficiency syndrome. J. Neurochem. 66:296‐302.
   Sei, Y., Whitesell, L., Kustova, Y., Paul, I.A., Morse III, H.C., Skolnick, P., and Basile, A.S. 1996b. Altered brain FYN kinase in a murine acquired immunodeficiency syndrome. F.A.S.E.B. J. 10:339‐344.
   Sei, Y., Kustova, Y., Li, Y., Morse III, H.C., Skolnick, P., and Basile, A.S. 1998. The encephalopathy associated with murine acquired immunodeficiency syndrome. Ann. N.Y. Acad. Sci. 840:822‐834.
   Spencer, D.C. and Price, R.W. 1992. Human immunodeficiency virus and the central nervous system. Ann. Rev. Microbiol. 46:655‐693.
   Toggas, S.M., Masliah, E., Rockenstein, E.M., Rall, G.F., Abraham, C.R., and Mucke, L. 1994. Central nervous system damage produced by expression of the HIV‐1 coat protein gp120 in transgenic mice. Nature 367:188‐193.
   Tyor, W.R., Power, C., Gendelman, H.E., and Markham, R.B. 1993. A model of human immunodeficiency virus encephalitis in SCID mice. Proc. Natl. Acad. Sci. U.S.A. 90:8658‐8662.
Key References
   Hartley et al., 1989. See above.
  Describes the characteristics of the infection and immunodeficiency, and indicates susceptible mouse strains. This paper allowed for the full utilization of LP‐BM5‐infected mice as a model of immunodeficiency diseases.
   Sei et al., 1998. See above.
  First published evidence that LP‐BM5‐infected mice also develop a neuropathology (spatial learning and memory deficit) as part of their immunodeficiency, as observed in a subset of HIV‐1 infections in humans. This behavioral assessment formed the basis for subsequent neurochemical and histological characterizations of these mice as models of AIDS dementia complex.
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