Light and electron microscopic studies of the 139A-H strain of scrapie passaged in hamsters

Introduction

We report here the light and electron microscopic neuropathology of the 139A-H strain of scrapie passaged in Syrian golden hamsters. Scrapie is a natural disease of sheep, goats and mouflons. It is a member of the group of transmissible spongiform encephalopathies or “prion diseases” [28,29]. Several experimental models in laboratory and transgenic animals have been described and several strains of the scrapie agent exist; the most widely experimentally used are the 263K (237sc) strains. The 139A-H strain is less widely used and thus deserves a more detailed description.

Material and methods

The 139A-H strain of scrapie

This strain was derived from the Scrapie Sheep Brain Pool (SSBP)-1 lineage [3,5]. Briefly, scrapie from a natural source was passaged through Cheviot sheep, Welsh Mountain sheep, goats, mice and led to the isolation of the “Chandler” scrapie strain [3]. The clone 139A strain was derived following the passage in hamsters [11] and this strain was made available to us by Dr. Richard I Carp, IBR, Staten Island, USA. Of interest, the distinct nature of the 139A-H strain was recently proved by the extended cell panel assay [26].

Electron microscopy

Two hamsters in the terminal stage of 139A-H scrapie infection and control sham-inoculated hamsters at the same interval after inoculation were anaesthetized with ketamine. They were perfused by intracardiac injection with saline followed by 150 ml of 1.25% glutaraldehyde and 1% paraformaldehyde prepared in cacodylate buffer (pH 7.4) and then by 50 ml of 5% glutaraldehyde and 4% paraformaldehyde.
Perfused animal carcasses were held at 4°C for at least 2 hours, after which brains were removed and several 1-mm3 samples were dissected under a bino­cular microscope from the parietal cortex, corpus callosum, CA2 region of the hippocampus, thalamus, cerebellum and brain stem. These samples were postfixed in 1% osmium tetroxide for 1-2 hours, dehydrated through a series of graded ethanols and propylene oxide, and then embedded in Epon resin (Serva). Semi-thin sections were stained with toluidine blue, blocks trimmed, and ultrathin sections stained with lead citrate and uranyl acetate. Sections were examined using a JEM 100 C transmission electron microscope.

Immunohistochemistry

For immunohistochemical staining, 4% formalin-fixed and paraffin-embedded hamster brains were used. Paraffin-embedded tissue samples were cut into 4-µm sections and processed routinely. The following antibodies were used: prion protein (PrP) 6H4 (1 : 400, mouse monoclonal, Prionics, Switzerland), glial fibrillary acidic protein (GFAP) (1 : 750, rabbit polyclonal, Dako, Denmark). Antigens were unmasked by incubating the sections in pH 6.0 citrate buffer using an autoclave (for PrP 6H4 only, 15 minutes, 121°C) or using proteinase K digestion of sections for 5 minutes (for GFAP only). Additionally, tissue sections for PrP 6H4 staining were incubated in 98-100% formic acid. For the detection of antibodies the EnVision + System-HRP with DAB as a chromogen (K4007 for use with mouse primary antibody, K4011 for use with rabbit primary antibody; Dako) was used according to the instructions of the manufacturer. Nuclei were stained with hematoxylin.

Analysis of PrP immunostaining

The intensity of the immunostaining were quantified in a single hamster brain using the images obtained from the AxioScan.Z1 system (Zeiss, Germany) using the Zeiss Zen 2.3 (Blue Edition) Software. The image analysis program Zen 2.3 (Blue Edition; Zeiss, Germany) – for light microscope image reconstruction was used. The recognition of the regions of interest was achieved with the 4× magnification lens and measures of intensity of staining were performed at 20× magnification (Figs. 1 and 2). To extract background from the pictures in the studied test areas, the neighboring area without the brain tissue was measured and obtained results of the mean value of this area were subtracted from the intensity of brain tissue. Arbitrary units of immunostaining intensity were used for comparative analysis after correction for background intensity.

Confocal laser microscopy

Additionally, double immunofluorescent stainings for confocal laser microscopy were performed. For double immunofluorescence stainings, 4% formalin fixed and paraffin-embedded hamster brains were used. Sections (4 µm) were mounted on tissue slides and processed routinely. The sections were treated with a citrate antigen retrieval solution. The following antibodies were used and combinations were applied: prion protein PrP 6H4 (1 : 400, Prionics, Switzerland), MAP-LC3 (1 : 2500, rabbit polyclonal, MBL). Tissue slides were mounted with Prolong Gold antifade reagent with DAPI (P36935, Molecular Probes). The staining results were evaluated with confocal laser-scanning microscope (Olympus FV1200, Japan). Serial optical sections in the Z dimension were captured to allow for three-dimensional (3D) reconstruction. Imaris software version 8.1.2 (Bitplane AG®, Zurich, Switzerland) was used to generate the 3D reconstructed images. Confocal Z-stacks comprising up to 20 images were reconstructed into 3D animations. Solid skinned cell-surface rendering was using Surface function. The filament tracer mode was used for detection of cell processes.

Results

The general neuropathological picture consisted of spongiform change (Fig. 3) and severe astrocytic gliosis (Fig. 4).

Topography of PrP

The topography of PrP deposits (in arbitrary units) is shown in Table I.

Electron microscopy

Spongiform vacuoles (Fig. 5) – these are always membrane bound and contain secondary vacuoles (i.e. membrane-bound compartments or vesicles within vacuoles) and curled membraned fragments.
Tubulovesicular structures (TVS) (Fig. 6) – these are vesicular structures of approximate 27 nm in diameter within neuronal processes – i.e. axonal terminal or dendrites. TVS are smaller and of higher electron density than synaptic vesicles. The significance of TVS remains unknown.
Dystrophic neurites (Figs. 7-9). Dendrites or axonal preterminals and terminals filled with electron-dense bodies, including small autophagic vacuoles. Apoptotic cell nuclei (Fig. 10). “Whorls”, concentric arrays of membranes were visible. A significance of those structures is unknown (Figs. 11-13).

Confocal laser microscopy

Double immunofluorescence stainings showed dot-like immunoreactivity of microtubule-associated protein (LC3) in the cell bodies while axons more often showed a fibrillar pattern. Interestingly, some neuronal processes showed only the neurofilament protein and LC3 positive structures were depleted of NFP immunoreactivity (Fig. 14).

Discussion

We characterized here the 139A-H scrapie strain using electron microscopy and immunohistochemistry. The topography of deposition of PrPSc revealed marked differences from region to region; the highest signal was observed in the CA2-molecular layer, CA1-pyramidal and entorhinal cortex. There are no data available to make a comparison [31]. The electron microscopy revealed extensive autophagy and, to a lesser degree, apoptosis.

Cell death in prion diseases

Data on autophagy in TSEs are scanty. Our initial experimental approach using the hamsters-adapted 263K or 22C-H strains of scrapie [14,19,20] was subsequently extended by studies of human brain biopsies from patients with sporadic CJD, variant CJD, and FFI [21] and mice infected with Gerstmann-Sträussler-Scheinker disease [18]. Autophagic vacuoles are defined as areas of the cytoplasm sequestered within double or multiple membranes (phagophores) of unknown origin [12]. In a classic Lampert [13] paper, a description of “dystrophic”, “regenerative” and “degenerative” neuritis was provided based on their appearance on transmission electron microscopy. Subsequently, it appeared that the criteria for separation into those 3 classes of neurites are not that clear and we used the term “dystrophic neurites” to cover all classes of altered neuronal branches that accumulate, irrespective of the cause, diverse subcellular organelles. Recently, Nixon et al. [2,25] interpreted many of those dense-bodies as small autophagic vacuoles. That dystrophic neurites in prion diseases are caused by the impairment of axonal transport was suggested many years ago by Gajdusek [6] and we showed it experimentally in experimental scrapie [7,23,24], BSE [22] CJD [16] and GSS [18] and, finally in chronic wasting disease [8]. Collectively, dystrophic neurites form a constant alteration in the TSE-affected brains. However, the mechanism(s) of their formation may only be speculated upon. We and others have suggested that neurons degenerate in prion diseases by autophagy [1,9,14,15,17,18,27,32], a process involving the removal of misfolded proteins and certain organelles, like mitochondria [4,10,30], and it was even suggested that spongiform vacuoles derived from giant autophagic vacuoles. Furthermore, the different dense bodies encountered in dystrophic neurites are in fact, autophagic vacuoles. In addition, multivesicular bodies are an ultrastructural hallmark of prion diseases [20].

Confocal laser microscopy

Dot-like immunoreactivity of LC3 is considered a marker of autophagy. In confocal laser microscopy of 139A-HA scrapie infected hamster brains, autophagic vacuoles were observed in some cell bodies, but most long neuronal processes showed mainly a fibrillar pattern of immunoexpression which is typical for undamaged axons. These findings are in accordance with the ultrastructural observation of autophagy in cell bodies and axonal terminals.

Disclosure

The authors report no conflict of interest.

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