Apoptotic cells, including macrophages, are prominent in Theiler's virus-induced inflammatory, demyelinating lesions - PubMed (original) (raw)

Apoptotic cells, including macrophages, are prominent in Theiler's virus-induced inflammatory, demyelinating lesions

Brian P Schlitt et al. J Virol. 2003 Apr.

Abstract

Theiler's murine encephalomyelitis virus (TMEV) persists in the mouse central nervous system principally in macrophages, and infected macrophages in culture undergo apoptosis. We have detected abundant apoptotic cells in perivascular cuffs and inflammatory, demyelinating lesions of SJL mice chronically infected with TMEV. T cells comprised 74% of apoptotic cells, while 8% were macrophages, 0.6% were astrocytes, and approximately 17% remained unidentified. In situ hybridization revealed viral RNA in approximately 1% of apoptotic cells.

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Figures

FIG. 1.

FIG. 1.

(A) Number of apoptotic cells determined by TUNEL assay in spinal cord sections of BeAn virus-infected SJL mice during active demyelinating disease between days 31 and 99. Apoptotic cells in the leptomeninges were excluded from analysis. Shown are mean numbers (○) of TUNEL-positive cells in each spinal cord segment (four to six sections per segment) from mice sacrificed on the indicated days. Bars indicate the mean number of apoptotic cells for each mouse. (B) Number of apoptotic cells (means ± SEs) in different spinal cord locations determined by TUNEL assay in sections of BeAn virus-infected SJL mice during active demyelinating disease between days 31 and 99 (same mice as in panel A).

FIG. 2.

FIG. 2.

Coronal spinal cord sections from a control mouse (A) and a BeAn virus-infected mouse sacrificed on day 69 (B to E). (B) Luxol fast blue staining reveals loss of myelin in the lateral and anterior columns; intense inflammatory infiltrates are visible in the leptomeninges in the ventral root entry zone and anterior commissure even at low-power magnification (×50). (C) Replicate section of panel B stained for TUNEL (no counterstaining), showing TUNEL-positive cells distributed in the white matter, especially anteriolaterally (magnification, ×50). (D) Magnification of the area boxed in panel C, showing distribution of TUNEL-positive cells around the periphery of a demyelinated area (compare with panel B). Arrows indicate TUNEL-positive cells in the pia layers of the leptomeninges (magnification, ×200). (E) Higher magnification (×400) of panel D, showing some TUNEL-positive cells exhibiting characteristic nuclear condensation indicative of apoptosis (arrows).

FIG. 3.

FIG. 3.

Two activated caspase-3 (blue)- and TUNEL (brown)-stained cord sections from a BeAn virus-infected SJL mouse. Right panel, approximately 20 caspase-3-positive cells; left panel, about 25 caspase-3-positive cells in the white matter and leptomeninges; asterisks in both panels indicate several more prominently TUNEL-stained cells in the white matter (magnification, ×400). No caspase-3 staining was detected in sections from control mice (data not shown).

FIG. 4.

FIG. 4.

Confocal images viewed with an Olympus IMT-2 microscope with digital deconvolution software of frozen spinal cord sections stained for nicked DNA (TUNEL, red); intact nuclei (4′,6′-diamidino-2-phenylindole, blue); and MOMA-2 (A), CD3+ (B), and GFAP (C) fluorescein isothiocyanate-labeled antibodies (green) and light microscopy image of a representative paraffin section taken from a BeAn virus-infected spinal cord on day 63 analyzed by combined in situ hybridization for BeAn virus RNA and TUNEL staining (arrows) for apoptosis (D). Colocalized nuclear and nicked DNA in cells resulted in a yellow-orange color (one cell in panels A and C and four cells in panel B). For combined in situ hybridization for BeAn virus RNA and TUNEL staining (arrows) for apoptosis, antisense and sense 35S-UTP-labeled BeAn virus probes (nucleotides 5967 to 6287) were synthesized with T7 and SP6 polymerase; probes with a specific activity of 6 × 107 cpm/μg in hybridization buffer were applied to tissue sections (D). In panel D, several cells (clustered together) on the extreme left have high viral RNA copy numbers (heavily grained area) and two separate cells on the lower right have low viral RNA copy numbers (asterisks). Only one infected, apoptotic cell, which is the bottommost cell in the cluster on the extreme left, is TUNEL positive (arrow; magnification, ×400). The insert shows the entire coronal spinal cord section from which the higher-power magnification field is taken; note grains over many cells in the anterior and lateral columns indicating the high level of viral persistence in this section (magnification, ×50).

FIG. 5.

FIG. 5.

Frequency of double-stained (antibodies to MOMA-2, CD3, or GFAP and to TUNEL) apoptotic cells in frozen spinal cord sections of four BeAn virus-infected SJL mice during persistent infection. The mean number (± SE) of double-positive cells identified as macrophages, T lymphocytes, and astrocytes per mouse is shown. In some instances, error bars were below graphic resolution.

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