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Deposition pattern and subcellular distribution of disease-associated prion protein in cerebellar organotypic slice cultures infected with scrapie

View Article: PubMed Central

ABSTRACT

Organotypic cerebellar slices represent a suitable model for characterizing and manipulating prion replication in complex cell environments. Organotypic slices recapitulate prion pathology and are amenable to drug testing in the absence of a blood-brain-barrier. So far, the cellular and subcellular distribution of disease-specific prion protein in organotypic slices is unclear. Here we report the simultaneous detection of disease-specific prion protein and central nervous system markers in wild-type mouse cerebellar slices infected with mouse-adapted prion strain 22L. The disease-specific prion protein distribution profile in slices closely resembles that in vivo, demonstrating granular spot like deposition predominately in the molecular and Purkinje cell layers. Double immunostaining identified abnormal prion protein in the neuropil and associated with neurons, astrocytes and microglia, but absence in Purkinje cells. The established protocol for the simultaneous immunohistochemical detection of disease-specific prion protein and cellular markers enables detailed analysis of prion replication and drug efficacy in an ex vivo model of the central nervous system.

No MeSH data available.


Neuropathological changes in 22L infected cerebellar slices. Slices exposed to Mock or 22L brain homogenate were cultured for 9 weeks. PrPd was detected following antigen denaturation using mAb 4H11 (green). (A) Neurons were stained with pAb anti-β-3-tubulin (red). (B) Purkinje cells were labeled with mAb against calbindin (red). (C) PAb anti-GFAP was used to stain astrocytes (red). (D) Microglia were detected with pAb anti-iba-1 (red). Nuclei were counterstained with Hoechst. Molecular and Purkinje cell layers of folia IX were imaged by confocal microscopy using the tile scanning function with identical imaging settings for each sample group. Scale bar: 50 μm.
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Figure 5: Neuropathological changes in 22L infected cerebellar slices. Slices exposed to Mock or 22L brain homogenate were cultured for 9 weeks. PrPd was detected following antigen denaturation using mAb 4H11 (green). (A) Neurons were stained with pAb anti-β-3-tubulin (red). (B) Purkinje cells were labeled with mAb against calbindin (red). (C) PAb anti-GFAP was used to stain astrocytes (red). (D) Microglia were detected with pAb anti-iba-1 (red). Nuclei were counterstained with Hoechst. Molecular and Purkinje cell layers of folia IX were imaged by confocal microscopy using the tile scanning function with identical imaging settings for each sample group. Scale bar: 50 μm.

Mentions: Consecutive sections of 22L infected and Mock exposed slices 9 weeks p.i. were imaged for vacuole formation (Figure 4). Consistent with in vivo vacuolization patterns (Kim et al., 1990a), spongiform changes were also apparent in the gray matter, particularly in molecular and Purkinje cell layers of 22L infected organotypic cerebellar slices. No vacuolation was observed in Mock exposed slices imaged using the same settings (data not shown). Confocal immunofluorescence analysis of 22L infected slices 9 weeks p.i. (~10 μm from the top of the slice) demonstrated a substantial reduction in β-3-tubulin immunoreactivity compared to Mock exposed slices, indicative of neuronal loss (Figure 5A). Numbers of Purkinje cells appeared reduced upon prion infection (Figure 5B). Purkinje cells in Mock exposed slices exhibited a well-defined arborization penetrating into the molecular layer of the cerebellum. By contrast, arborizations of Purkinje cells in 22L infected slices appeared degenerated. An increase in GFAP immunoreactivity was observed in prion infected compared to Mock exposed slices (Figure 5C). Concomitantly, the number of iba-1 positive microglia increased in 22L infected slices (Figure 5D). In summary, characteristic hallmarks of prion diseases including spongiform changes, Purkinje cell degeneration, astro- and microgliosis were reproduced in 22L infected organotypic cerebellar slices from C57BL/6JRj mice.


Deposition pattern and subcellular distribution of disease-associated prion protein in cerebellar organotypic slice cultures infected with scrapie
Neuropathological changes in 22L infected cerebellar slices. Slices exposed to Mock or 22L brain homogenate were cultured for 9 weeks. PrPd was detected following antigen denaturation using mAb 4H11 (green). (A) Neurons were stained with pAb anti-β-3-tubulin (red). (B) Purkinje cells were labeled with mAb against calbindin (red). (C) PAb anti-GFAP was used to stain astrocytes (red). (D) Microglia were detected with pAb anti-iba-1 (red). Nuclei were counterstained with Hoechst. Molecular and Purkinje cell layers of folia IX were imaged by confocal microscopy using the tile scanning function with identical imaging settings for each sample group. Scale bar: 50 μm.
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Figure 5: Neuropathological changes in 22L infected cerebellar slices. Slices exposed to Mock or 22L brain homogenate were cultured for 9 weeks. PrPd was detected following antigen denaturation using mAb 4H11 (green). (A) Neurons were stained with pAb anti-β-3-tubulin (red). (B) Purkinje cells were labeled with mAb against calbindin (red). (C) PAb anti-GFAP was used to stain astrocytes (red). (D) Microglia were detected with pAb anti-iba-1 (red). Nuclei were counterstained with Hoechst. Molecular and Purkinje cell layers of folia IX were imaged by confocal microscopy using the tile scanning function with identical imaging settings for each sample group. Scale bar: 50 μm.
Mentions: Consecutive sections of 22L infected and Mock exposed slices 9 weeks p.i. were imaged for vacuole formation (Figure 4). Consistent with in vivo vacuolization patterns (Kim et al., 1990a), spongiform changes were also apparent in the gray matter, particularly in molecular and Purkinje cell layers of 22L infected organotypic cerebellar slices. No vacuolation was observed in Mock exposed slices imaged using the same settings (data not shown). Confocal immunofluorescence analysis of 22L infected slices 9 weeks p.i. (~10 μm from the top of the slice) demonstrated a substantial reduction in β-3-tubulin immunoreactivity compared to Mock exposed slices, indicative of neuronal loss (Figure 5A). Numbers of Purkinje cells appeared reduced upon prion infection (Figure 5B). Purkinje cells in Mock exposed slices exhibited a well-defined arborization penetrating into the molecular layer of the cerebellum. By contrast, arborizations of Purkinje cells in 22L infected slices appeared degenerated. An increase in GFAP immunoreactivity was observed in prion infected compared to Mock exposed slices (Figure 5C). Concomitantly, the number of iba-1 positive microglia increased in 22L infected slices (Figure 5D). In summary, characteristic hallmarks of prion diseases including spongiform changes, Purkinje cell degeneration, astro- and microgliosis were reproduced in 22L infected organotypic cerebellar slices from C57BL/6JRj mice.

View Article: PubMed Central

ABSTRACT

Organotypic cerebellar slices represent a suitable model for characterizing and manipulating prion replication in complex cell environments. Organotypic slices recapitulate prion pathology and are amenable to drug testing in the absence of a blood-brain-barrier. So far, the cellular and subcellular distribution of disease-specific prion protein in organotypic slices is unclear. Here we report the simultaneous detection of disease-specific prion protein and central nervous system markers in wild-type mouse cerebellar slices infected with mouse-adapted prion strain 22L. The disease-specific prion protein distribution profile in slices closely resembles that in vivo, demonstrating granular spot like deposition predominately in the molecular and Purkinje cell layers. Double immunostaining identified abnormal prion protein in the neuropil and associated with neurons, astrocytes and microglia, but absence in Purkinje cells. The established protocol for the simultaneous immunohistochemical detection of disease-specific prion protein and cellular markers enables detailed analysis of prion replication and drug efficacy in an ex vivo model of the central nervous system.

No MeSH data available.