Reflections on Cerebellar Neuropathology in Classical Scrapie

Abstract

In this review, the most important neuropathological changes found in the cerebella of sheep affected by classical natural scrapie are discussed. This disease is the oldest known of a group of unconventional "infections" caused by toxic prions of different origins. Scrapie is currently considered a "transmissible spongiform encephalopathy" (due to its neuropathological characteristics and its transmission), which is the paradigm of prion pathologies as well as many encephalopathies (prion-like) that present aberrant deposits of insoluble protein with neurotoxic effects due to errors in their catabolization ("misfolding protein diseases"). The study of this disease is, therefore, of great relevance. Our work data from the authors' previous publications as well as other research in the field. The four most important types of neuropathological changes are neuron abnormalities and loss, neurogliosis, tissue vacuolization (spongiosis) and pathological or abnormal prion protein (PrP) deposits/deposition. These findings were analyzed and compared to other neuropathologies. Various aspects related to the presentation and progression of the disease, the involution of different neuronal types, the neuroglial responses and the appearance of abnormal PrP deposits are discussed. The most important points of controversy in scrapie neuropathology are presented.

Keywords: Purkinje cells; abnormal PrP deposits/deposition; astrogliosis; calbindin immunoreactivity; calretinin immunoreactivity; cerebellum; classical natural scrapie; microgliosis; spongiosis.

Figure 1

Main neuropathological alterations in classical scrapie. (A) Neuronal changes and loss. In these cerebellar folia, Purkinje neurons of different morphologies are observed: normal, hypertrophic (black arrow), atrophic (green arrows) and dystrophic (blue arrow). Likewise, areas exist where neurons have been lost (white arrows). Calbindin immunostaining plus hematoxylin contrast. (B) “Spongiform” (vacuolar) structures. Vesicles of different types are seen both in the neuropil (green arrow) and in the cytoplasm of neurons (white arrows). Calbindin immunostaining plus hematoxylin contrast. (C,D) Neuroglial reactivity. In (C), microgliosis is shown (proliferation of different types of microglia—rounded or branched). LN3 immunostaining plus Ni contrast. In (D), astrogliosis (hypertrophy, hyperplasia and increased number of gliofibrils) is shown. GFAP immunostaining plus hematoxylin contrast. (D) Abnormal prion protein (PrP) deposits/deposition. Various types of accumulations in the neuropil associated with cells, both in the form of dispersed or coalescent granulations and in a star-like shape. Abnormal PrP immunostaining plus hematoxylin contrast. (E) Abnormal PrP immunostaining plus hematoxylin contrast. (Bar: A = 125 µm; B = 20 µm; C = 125 µm; D = 30 µm; E = 35 µm).

Figure 2

Calbindin-immunopositive Purkinje cells in control (A) and scrapie-affected sheep (B–I). (B) Cells of normal appearance in a case of preclinical scrapie, with a similar pattern to the control cases. (C) Cells of different appearance (normal, hypertrophic, dystrophic with rarefaction of their cytoplasm) in a case of clinical scrapie. (D,E) Cells of very different immunoreactivity in two cases of terminal scrapie. (F) Cytoplasmic rarefaction of the basal cytoplasm in an immunopositive cell of clinical scrapie. (G) Intense vacuolization in a positive cell. (H,I) Hypertrophic/hyperreactive cells. In (H), the hypertrophy of the initial dendrites is shown, and in (I), the hypertrophy of the distal dendrites that reach the pia mater. (A–F), hematoxylin contrast; (H,I), without contrast. (Bar: A, B = 125 µm; C–E = 25 µm; F, G = 20 µm; H,I = 30 µm).

Figure 3

Electron microscopy images of the basal region of non-normal Purkinje neurons. Scrapie sheep in clinical phase. (A) Dystrophic cell with cytoplasmic rarefaction (dissolution/loss of subcellular organelles). Not defined debris are observed. Tubulo-vesicular formations appear (more notable in the upper left part of the image) as well as other electrodense microvesicles. Insert: magnification similar to (B) for comparison. (B) Hypertrophic cell (diameter, 42 µm) with a great abundance of subcellular organelles, although rough endoplasmic reticulum (RER) is more disorganized. Selected cytoplasmic regions are similar of those control neurons, but others resemble what is seen in dystrophic neurons (Bar: A = 0.5 µm; B = 3 µm).

Figure 4

Calretinin-immunopositive cells in the cerebellar cortex of scrapie sheep (hematoxylin contrast). (A–C) Lobe VI (neocerebellum). In A, a slight reaction in Purkinje cells (PCs) and a greater intensity in some Golgi and brush cells of the granule cell layer as well as positivity in some parallel fibers are observed in a case of preclinical scrapie, a pattern similar to that shown by healthy controls. In (B), a case of clinical scrapie, the reaction pattern is maintained, although the intensity of the reaction is greater in some Purkinje neurons and cells of the granule cell layer (in the image, a Lugaro cell). In (C), a case of terminal scrapie, a very intense reaction is seen in hypertrophic Golgi cells of the granule cell layer (a vacuole is surrounded by its dendrites). The number of immunopositive Purkinje cells appears to decrease. (D–G) Lobe X (archicerebellum). In (D) and (E), a great diversity of reactions is observed in Purkinje cells (from negative to slightly positive in (D) to strongly positive in (E) as well as an intense reactivity in Golgi and brush cells of the granule cell layer. (The cell density of these cell types is 3–5 times higher than those observed in the neocerebellum). The reaction pattern is the same as that shown in control cases. In terminal cases (F), it seems to increase the reactivity in Purkinje cells (some of them are very hyperreactive) and decrease the intensity of the reaction in cells of the granule cell layer. (G) Detail of hyperreactive hypertrophic Purkinje cells in a case of clinical scrapie. (H) Hypertrophic/hyperreactive PCs in clinical phase. (I) Thin calretinin-positive varicose fibers that are prolongations of astrocytes of the granule cell layer to the molecular layer (“Weigert fibers”), observable only in clinical and terminal cases. (J) Detail of the elongation of monopolar dendrites and axons of brush cells that form loops in their path between grains and vesicles. Clinical scrapie case. (Bar: A = 40 µm; B = 25 µm; C–G = 40 µm; H–J = 20 µm).

Figure 5

Astrogliosis in the cerebellar cortex of preclinical (A), clinical (B–F) and terminal (G) scrapie cases (GFAP immunostaining plus hematoxylin contrast). In the preclinical stage, the astroglial pattern is similar to that observed in controls. In the clinical stage, greater astrogliosis is observed, which can present different characteristics in different folia. In (B) and (C), there is hyperreactivity and hypertrophy in the astrocytes of the granular layer and in the Bergmann fibers of the molecular layer. In (D), great hypertrophy of these fibers is shown. (E) and (F) show greater astrogliosis in the basal area of the Purkinje layer. (G) Astrogliosis in a case of terminal scrapie. (H,I). Parallel GFAP-immunostained (H) and abnormal PrP-immunostained (I) sections, respectively. There is a coincidence of granular deposits of abnormal PrP deposits/deposition in the Purkinje layer and the granule cell layer, but the star-shaped deposits in the molecular layer do not correspond to GFAP-immunopositive stellate astrocytes in the molecular layer. (Bar: A = 50 µm; B–E = 40 µm; F,G = 25 µm; H,I = 50 µm).

Figure 6

Abnormal PrP deposits/deposition (abnormal PrP immunoreaction—L42 1/500; Bio-Pharm, Darmstadt, Germany—plus hematoxylin contrast). (A) Deposits in a sheep in the preclinical phase of scrapie. Slight deposits are observed only in some folia in the layers of the cerebellar cortex. (B,C) Star-like deposits in the molecular layer (in B) and various types of deposits in all layers in two cases of sheep with infection in the clinical stage. (D) Diffuse granular deposits in all layers of a terminal-phase sheep case. Star-like deposits do not appear. (E–G) Details of prion deposits in clinical phase. In (E), star-like deposits are seen, which suggest association with non-GFAP-immunopositive astroglial cells. Hematoxylin-stained glial nuclei are seen in some of these deposits. In (F), a high degree of coalescence of the granular deposits is observed in the basal area of the Purkinje layer, similar to dense formations of astroglial processes in these areas (see Figure 4). In (G), a large accumulation of granular prion deposition is observed surrounding the areas of dystrophic Purkinje cells (with rarefied cytoplasm), but without the appearance of abnormal PrP immunoreaction in these cells. Abnormal PrP immunoreaction with hematoxylin contrast (in different concentrations). (Bar: A–D = 40 µm; E = 10 µm; F = 25 µm; G = 10 µm).

Figure 7

Electron microscopy images of vacuoles (“spongiosis”). (A) Large vacuole in a Purkinje cell without an apparent membrane. There seems to be an evagination (arrow, lower left area) towards the surface of the neuron. (B) Large (*) and small (**) vacuoles in the neuropil of the molecular layer surrounded by an astroglial process. Vacuole membranes are more or less apparent. (C) Large vacuole (*) in the neuropil of the molecular layer surrounded by a thin Purkinje cell dendrite (arrow), defined by accumulation of hypolemmal cisterns. (B) and (C). Section normal to parallel fibers. (D) Large vacuole surrounding a Purkinje dendrite that presents a large accumulation of hypolemmal cisterns but retains synaptic connections with climbing fiber terminals (arrow). (E) Various types of medium-sized vesicles surrounding Purkinje cell dendrites (*). Some vesicles lack cellular debris in their interior, but others retain cellular organelle debris (arrow). The latter may be vacuolized Bergmann fibers. (F) Hypertrophic astroglia cell with different types of vacuoles and intracellular dense bodies. (Bar: A,C,D = 3.5 µm; B and F = 1.5 µm).

Figure 8

Electron microscopy images of vacuoles and subcellular bodies. (A) Dystrophic dendrites of stellate cells of the molecular layer (*), located between parallel fibers, showing autophagic vacuoles. (B) Intracellular vacuole without a defined membrane at an axon terminal of a basket cell on a Purkinje neuron (*). (C) “Tubulovesicular” formations with translucent content in association with electron-dense vesicles. (D) Cytoplasm of dystrophic Purkinje cells with loss of organoids, hypertrophic hypolemmal cisterns and microvesicles (arrow). (E) Purkinje atrophic cells surrounded by extraneuronal vesicles (*). They have a dense cytoplasm (“dark neurons”) with a large number of dense vesicular structures that are more or less complex. (Bar: A = 3.5 µm; B = 0.5 µm; C = 30 nm; D = 0.5 µm; E = 1 µm).