The Paraffin-Embedded Tissue Blot Detects PrPSc Early in the Incubation Time in Prion Diseases

Abstract

With the appearance of bovine spongiform encephalopathy (BSE) and a new variant of Creutzfeldt-Jakob disease (nvCJD) that seems to be caused by BSE, there is an increased need for improvement of diagnostic techniques and recognition of all variants of prion diseases in humans and animals. Publications on the immunohistochemical identification of PrP(Sc) in the tonsils and appendix in the incubation period of nvCJD indicate that new and more sensitive techniques for the detection of PrP(Sc) in various tissues may be a valuable tool for early diagnosis in prion diseases. We developed a new and sensitive technique to detect PrP(Sc) in formalin-fixed and paraffin-embedded tissue, the paraffin-embedded tissue blot (PET blot), and reinvestigated archival brain material from CJD as well as BSE and scrapie. In addition, C57/Bl6 mice experimentally infected with the ME7 strain were investigated sequentially during the incubation time to compare this new technique with conventional methodologies. The PET blot detects PrP(Sc) in idiopathic (sporadic) and acquired prion diseases, even in cases with equivocal or negative immunohistochemistry, and is more sensitive than the conventional Western blot and histoblot techniques. The PET blot makes possible the detection of PrP(Sc) during the incubation period long before the onset of clinical disease and in prion disease variants with very low levels of PrP(Sc). In mice experimentally infected with the ME7 strain, the PET blot detects PrP(Sc) in the brain 30 days after intracerebral inoculation-145 days before the onset of clinical signs. Its anatomical resolution is superior to that of the histoblot technique. It may therefore be of particular interest in biopsy diagnosis. Thus it complements other tissue-based techniques for the diagnosis of prion diseases in humans and animals.

Role of Interleukin-1 in Prion Disease-Associated Astrocyte Activation

“…The accumulation of the disease-associated isoform of the prion protein PrP Sc in IL-1RI Ϫ/Ϫ and C57/B6 mice was examined by immunohistochemistry and the recently developed paraffin-embedded tissue blot (PET blot) technique. 29 Using conventional immunohistochemistry less stainable PrP Sc was detectable in the IL-1RI Ϫ/Ϫ mice than in the C57/B6 controls at 125 dpi ( Figure 5), but both groups appeared primarily similar at the terminal stage of the disease (data not shown). The PET blot technique facilitates the depiction of larger brain areas and allows for a stringent discrimination between normal PrP and proteinase K-resistant PrP Sc .…”

“…29 Briefly, 6-m paraffin-sections of formalinfixed brain tissues were transferred onto a nitrocellulose membrane and, after dewaxing, treated with proteinase K for digestion of normal PrP C . Remaining membranebound protein was subsequently denatured with 3 mol/L guanidine isothiocyanate.…”

Preclinical deposition of pathological prion protein PrPSc in muscles of hamsters orally exposed to scrapie

“…Brains were immediately removed from sacrificed hamsters and immersed for 48 hours in 4% phosphate-buffered formalin as described by Schulz-Schaeffer et al (39). After treatment with formic acid for 1 hour at room temperature, brains were placed in 4% phosphate-buffered formalin for a further 24 hours and subsequently trimmed coronally into 2-to 3-mm-thick slices.…”

“…After treatment with formic acid for 1 hour at room temperature, brains were placed in 4% phosphate-buffered formalin for a further 24 hours and subsequently trimmed coronally into 2-to 3-mm-thick slices. Samples were embedded in paraffin wax, and PET blotting was carried out as described (39), with slight modifications. Six-micrometer microtome slices were mounted onto nitrocellulose membranes and dried flat at 50°C for 16 hours.…”

Cell models of prion infection

“…Distribution of PrP res deposition in the infected brain, as assessed by PETblot or histoblot can vary in a strain-dependent manner [127,137], raising the possibility that distinct strains multiply in distinct brain cell types [36]. Since the central nervous system is extremely complex, the basis of prion identity and diversity should be advantageously investigated in single cell lines faithfully propagating several strains of prions.…”