Age-related changes in susceptibility of rat brain slice cultures including hippocampus to encephalomyocarditis virus

Abstract

Replication of the D variant of encephalomyocarditis virus (EMC-D) and its cytopathic effects were studied in the brain slice cultures including hippocampus (hippocampal slice) obtained from postnatal 1-, 4-, 7-, 14-, 28-and 56-day-old Fischer 344 rats. At 0, 12, 24, 36 and 48 h after infection, virus titres of the slices and culture media were assayed. Viral replication was observed in cultures from 1-to 28-day-old rats, and the highest titre was recorded in the slice and culture medium from the youngest rat. The peak of virus titre decreased with age and no distinct viral replication was observed in the cultures from 56-day-old rats. Light microscopy revealed that degenerative and necrotic changes appeared in the infected hippocampal slices from 1- to 28-day-old rats, and the changes became less prominent with age. In situ hybridization and indirect immunofluorescence staining showed that positive signals of viral RNA and antigen were prominent in younger rats and decreased with age. These results suggest that an age-related decrease in the susceptibility of rat brain to EMC-D is less related to the maturation of the immune system but possibly to that of the neurone.

Figure 1

Virus titres of (a) 1-day-old (b) 4-day-old (c) 7-day-old (d) 14-day-old (e) 28-day-old and (f) 56-day-old rat hippocampal slices (•) and culture media (○). The value and bar are expressed as arithmetic mean of triplicate wells ± SD.

Figure 2

Histopathology of hippocampal slices (CA1 region) from (a, e) 1-day-old, (b, f) 7-day-old, (c, g) 28-day-old and (d, h) 56-day-old rats. In EMC-D-uninfected slices (a-d), there is no pathological change. In infected slices (e-h), the degeneration and necrosis of neurones occur in 1- to 28-day-rats and become lighter with age, no changes in 56-day-old rats. CA1, hippocampal region; O, stratum oriens; R, stratum radiatum. H & E. Bar = 20μm.

Figure 3

Viral RNA distribution (CA1 region). In EMC-D-infected hippocampal slices from (a) 1-day-old, (b) 7-day-old, (c) 14-day-old, (d) 28-day-old and (e) 56-day-old rats, the intensity of positive signals of viral RNA (hybridized with antisense probe) decreases with age. In control sections from 1-day-old rat, (f) there are a few signals around neuroneic nuclei (hybridized with sense probe) in infected hippocampal slices and (g) no positive signals (hybridized with antisense probe) in uninfected hippocampal slices. ISH. Bars: (a–e), 50 μm; (f–g), 22 μm.

Figure 4

Viral antigen distribution (CA1 region). In EMC-D-infected hippocampal slices from (a) 1-day-old, (b) 7-day-old and (c) 56-day-old rats, there are many viral antigen-positive cells in the hippocampus from 1-day-old and 7-day-old rats, but hardly any viral antigen in 56-day-old rats. (d) The viral antigen is negative in uninfected hippocampal slices from 1-day-old rat. IFA. Bar = 28 μm.