Fig 1.
Detection of PrPSc by immunohistochemistry using monoclonal antibody D13 in thalamus at 40 and 60 days post-infection by microinjection with scrapie strains 22L (A,B), RML (C,D), or ME7 (E,F). (A) For strain 22L, at 40 dpi several areas of PrPSc deposition around apparent individual cells (arrow) and around blood vessels (arrowhead) were noted in dorsomedial (DM) region of the thalamus on the ipsilateral side. (B) At 60 dpi PrPSc was detected in the same areas but was more abundant than in (A). (C) After infection with RML, at 40 dpi small amounts of finely punctate PrPSc was barely visible (arrows) in clusters around large nuclei, but with magnification (inset) was easily seen in DM thalamus on ipsilateral side (arrows). (D) In contrast, at 60 dpi RML PrPSc deposits were widespread in DM thalamus (arrows) and were often on blood vessels (arrowhead), or in neuropil spaces between cells (zoom on figure to see details). (E) After ME7 infection, no PrPSc was seen in thalamus at 40 dpi, but a small amount of linear (axonal) punctate PrPSc was observed in the ipsilateral substantia nigra (inset, arrow). (F) At 60 dpi ME7 coarsely punctate PrPSc was noted in DM thalamus as amorphous aggregates of varying size (arrow). Occasional perineuronal PrPSc was also seen (arrowheads, zoom to see details). The area of ME7 PrPSc deposition was smaller than that for strains 22L and RML at this same time. Scale bar shown in panel A is 400 micrometers and applies to all panels.
Fig 2.
Western blots comparing PrPSc levels in thalamus at 40 and 60 dpi from three scrapie strains.
(A) 40 dpi. (B) 60 dpi. On each western blot, two representative thalamus samples are shown for each scrapie strain. Scrapie strains shown are indicated across the top of each western blot. Lanes 1–6 were loaded with 0.36 mg of brain equivalents. In both panels, lane 7 (Bl) indicates an intentional blank lane, and lane 8 (C) is a positive control thalamus from a 60 dpi 22L sample loaded with 0.24 mg brain equivalents. Western blots were probed with anti-PrP antibody D13 and exposed to film for 8 minutes. To avoid issues with pixel saturation, densitometry was performed on a 4 minute exposure to film. The adjusted volumes (immunoreactive band volume with global background subtracted) in arbitrary units are given below each lane. At both time points shown the mice inoculated with scrapie strain ME7 had significantly less detectable PrPSc (lanes 5&6) than was seen in samples from 22L- and RML-infected mice.
Fig 3.
Cell-type associations of PrPSc in thalamus after microinjection with scrapie strains 22L, 40 dpi (A-D), RML, 60 dpi (E-H) and ME7, 60 dpi (I-L). PrPSc was detected by IHC with monoclonal antibody D13, and slides were also dual-stained with a second antibody to detect neurons (NeuN), astroglia (GFAP), microglia (Iba1) and oligodendroglia (Ol2), as described in methods. (A) Several cells positive for both 22L PrPSc and GFAP (arrows) are noted. Inset shows a PrPSc-positive stellate cell at 20 dpi that was GFAP-negative. (B) 22L PrPSc-positive cells negative for Neu-N (arrows) are shown. Arrowhead shows PrPSc on a small blood vessel. Blue arrow shows PrPSc in neuropil not associated with any cell body. (C) One cell with 22L PrPSc and Iba1 co-association is shown (blue arrow). Several PrPSc-positive cells negative for Iba1 are also seen (black arrows). (D) Cells positive for 22L PrPSc and Olig2 (Ol2) (arrows) plus neuropil PrPSc (arrowhead) is seen. (E) RML PrPSc co-associates with GFAP-positive astroglia (arrows). (F) Intense RML PrPSc staining (arrows) is seen around Neu-N-negative cells with pale blue nuclei. Diffuse PrPSc staining is seen in neuropil background and is often adjacent to Neu-N-positive neurons, therefore possible association with neurons is difficult to exclude. (G) Lack of co-localization of RML PrPSc and Iba1. PrPSc-positive cells (arrows) are distinguishable from Iba1-positive cells (arrowhead). (H) Several cells are co-stained for RML PrPSc and Olig2 (arrows). Less dense PrPSc covers much of background neuropil. (I) After ME7 infection shows GFAP-positive astrocytes near larger PrPSc deposits (black arrows) in neuropil, but most astrocytes are PrPSc-negative (blue arrow). (J) Neu-N-positive neuron with ME7 PrPSc co-localization (arrow) on ipsilateral side. Additional PrPSc staining in larger aggregates (arrowheads) appears in neuropil. (K) No accumulation of ME7 PrPSc on Iba1-positive cells (blue arrow). PrPSc was seen in neuropil (black arrow) and possibly associated with large unstained neurons (arrowhead). (L) Large ME7 PrPSc aggregates in neuropil (arrows) with no evidence for accumulation on cells positive for Olig2 (arrowhead). Scale bar shown in panel A is 50 μm and applies to all panels.
Fig 4.
Cell-type associations of PrPSc in cortex, substantia nigra and hypothalamus after striatal microinjection of scrapie strains 22L (A-D), RML (E-H) or ME7 (I-L). PrPSc was detected by IHC with monoclonal antibody D13 (brown color), and some slides were also dual-stained with a second antibody to detect neurons (NeuN) or astroglia (GFAP) as described in methods. (A) At 40 dpi 22L PrPSc is associated with a stellate GFAP-positive astrocyte (arrow). Nearby larger neurons and vascular endothelial cells are PrPSc-negative. (B) At 40 dpi stellate cells (arrows), likely to be astrocytes, are positive for 22L PrPSc and are intermingled with neurons stained with anti-NeuN (pink). (C) At 40 dpi in substantia nigra 22L PrPSc is not associated with Neu-N-positive neurons (pink) but instead surrounds cells with small blue nuclei, possibly astrocytes. (D) In hypothalamus at 60 dpi single stained 22L PrPSc surrounds small cells with blue nuclei (arrows) and is less often adjacent to round neurons with prominent nucleoli. (E) At 60 dpi RML PrPSc is associated with a GFAP-positive astrocyte (arrow) and also with perivascular astrocyte surrounding a small blood vessel (arrowhead). (F) In cortex at 60 dpi RML PrPSc surrounds cells with small nuclei (arrows) near Neu-N-positive neurons, similar to 22L in panel (B). (G) In substantia nigra at 60 dpi RML PrPSc is associated with cell surface of two Neu-N-positive neurons (arrows). PrPSc is also seen in the neuropil between neurons (arrowhead). (H) At 80 dpi RML PrPSc in hypothalamus, detected by single staining with D13, is associated with neuropil (arrowheads), blood vessel (blue arrow) and probable astroglia with small nuclei (black arrow). (I) At 100 dpi ME7 PrPSc in cortex is deposited in fine and coarse aggregates in neuropil (arrowheads), but is not associated with GFAP-positive astroglia (arrow). (J) At 100 dpi in cortex ME7 PrPSc is associated with NeuN-positive neurons (arrows). Occasional coarse aggregates are also seen in the neuropil (arrowhead). (K) At 60 dpi in substantia nigra ME7 PrPSc surrounds some Neu-N-positive neurons and also is seen as punctate deposits (arrowheads) near neurons and as linear staining suggestive of axons. (L) At 100 dpi in hypothalamus ME7 PrPSc is shown using single staining in a perineuronal distribution (arrows) and also as coarse neuropil aggregates (arrowheads). Scale bar shown in panel A is 50 μm and applies to all panels.
Fig 5.
Transcript analysis of astroglial and microglial markers in thalamic RNA samples from mice infected with scrapie strains 22L, RML, or ME7.
(A) The ΔCT of Gfap, an indicator of astroglial activation (open circles), showed astrogliosis was significantly increased early in 22L-infected thalamus (40 dpi) and increased further at 60 and 80 dpi. Infection with RML reached a similar increase in Gfap by 60 dpi, whereas ME7-infected mice demonstrated a significant increase in Gfap similar to that of RML and 22L by 80 dpi. Likewise, the ΔCT of Gpr84, an indicator of microglial activation (closed circles), illustrated a temporal increase in thalamic expression during infection by all three scrapie strains tested, albeit delayed by 20 dpi relative to Gfap. The dashed horizontal line indicates the mean ΔCT of Gfap and the solid horizontal line indicates the mean Gpr84 ΔCT in mock infected mice. (B) The relatively unchanged expression of vimentin (Vim), an astrocyte associated gene, or the chemokine receptor Cx3cr1, a microglia associated gene, suggested the early transcriptional changes observed in the gliosis markers Gfap and Gpr84 in infected thalamus were not due to an overt expansion of either cell population. The dashed horizontal lines indicate the mean ΔCT in mock infected mice as a reference. The bars for all samples indicate the mean and 1 standard deviation. The ΔCT for each mouse RNA sample was calculated by subtracting the geometric mean CT of three housekeeping genes (Actin, Gapdh, and Hsp90ab1) from the CT of the gene of interest. Each dot represents an individual mouse.
Fig 6.
Inflammatory genes significantly increased in the thalamus of scrapie-infected mice.
The changes in transcription for 86 inflammatory cytokines and their receptors was analyzed by qRT-PCR and categorized by time of increase (S1 Table for complete list of genes tested). The 11 genes shown here demonstrated a significant increase in expression in either 22L-infected or RML-infected thalamus of mice at the earliest time assessed (40 dpi and 60 dpi, respectively). The solid horizontal line on each graph indicates the mean ΔCT in mock infected mice as a reference. The bars for all samples indicate the mean and 1 standard deviation. The ΔCT for each mouse RNA sample was calculated by subtracting the geometric mean CT of three housekeeping genes (Actin, Gapdh, and Hsp90ab1) from the CT of the gene of interest. Each dot represents an individual mouse.
Table 1.
Quantitative analysis by qRT-PCR of fold change of mRNA for specific genes in thalamus of mice microinjected with scrapie strain 22L at 40, 60 and 80 days post-injection.
Table 2.
Quantitative analysis by qRT-PCR of fold change of mRNA for specific genes in thalamus of mice microinjected with scrapie strain RML at 60, 80 and 100 days post-injection.
Table 3.
Quantitative analysis by qRT-PCR of fold change of mRNA for specific genes in thalamus of mice microinjected with scrapie strain ME7 at 60, 80 and 100 days post-injection.
Table 4.
Number of neuroinflammatory genes increased in 22L scrapie-infected mice at 80 dpi compared to mice infected with LaCrosse virus (LACV) at 5 dpi.
Table 5.
Number of neuroinflammatory genes increased in 22L scrapie-infected mice at 80 dpi compared to mice infected with BE retrovirus at 21–28 dpi.
Table 6.
Overview of number of mice per group in study of cellular accumulation of PrPSc by immunohistochemistry at various time-points after scrapie microinjection using three different scrapie strains.