PMC5769208_01
PMC5769208_02
The patient's Archimedes spirals (right arm), worsened over time - tremor rating = 2 (moderate) at age 85 (left panel) and tremor rating = 3 (severe) at age 93, which was 8.5 months prior to death (right panel)
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Fig1
During follow-up, his Archimedes spirals continued to show considerable tremor, which worsened over time - right/left ratings of 2/2 (age 85) evolved to 3/3 (age 93 [8.5 months prior to death]) (Fig. 1). An abundance of several other Purkinje cell axonal changes was identified by calbindinD28k immunohistochemistry (esp. axonal branching and axonal recurrent collaterals; Fig. 2g-i, Table 1), as well as an increase in Purkinje cell dendritic swellings on LH&E and Bielschowsky-stained sections (Table 1).
Degenerative changes in the patient's cerebellar cortex (b, c, e, f, h, i) compared with a control (a, d, g) with LH&E stain (a-c), Bielschowsky stain (d-f) and calbindinD28k immunostain (g-i). The molecular layer is thinner (b) and there is marked Purkinje cell loss with scattered, often atrophic Purkinje cells (b, c, white arrows) and Bergmann gliosis (b, c). Basket cell processes are coarse (e, yellow arrows), often around extant or atrophic (f, white arrow) Purkinje cells, and basket cell processes may be largely absent (e, yellow bars). Compared with control Purkinje cells (g), the patient's Purkinje cell dendritic arbors are often stunted (h, i) and axonal changes including torpedoes (c, f, h, i, large black arrows), axonal branching (H, small black arrows) and recurrent collaterals (h, i, carets) are increased. Scale bars: 100 mum, A, B, D, E; 50 mum, C, F; 100 mum, G-H
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Fig2
The cerebellar cortex was characterized by a range of degenerative changes (Fig. 2), as have been described in patients with ET. The loss of granular neurons was patchy, with a tendency to more severely affect the tips of folia than their depths. There was an increase in torpedoes on both LH&E and Bielschowsky-stained sections and marked hypertrophy of basket cell axonal processes along with areas where basket cell processes were absent (Fig. 2c-f; Table 1). An abundance of several other Purkinje cell axonal changes was identified by calbindinD28k immunohistochemistry (esp. axonal branching and axonal recurrent collaterals; Fig. 2g-i, Table 1), as well as an increase in Purkinje cell dendritic swellings on LH&E and Bielschowsky-stained sections (Table 1).
Degenerative changes in the patient's dentate nucleus (b, d, f, h) compared with a control (a, c, e, g) with LH&E stain (a-d), GAD immunostain (e-f) and calbindinD28k immunostain (g-h). a, b Myelin pallor in the patient's amiculum (a) and hilum (h) of the dentate nucleus. c, d Dentate neuronal density is normal, although the soma of these neurons was often atrophic (d). Marked reduction of afferent synaptic density from Purkinje cells in dentate demonstrated by GAD (e, f) and calbinidinD28k immunostain (g, h). GAD + -synaptic puncta around large GAD-negative dentate neurons (e, carets in bottom panel) are reduced (f), and some residual synaptic puncta are enlarged (F, small black arrow in bottom panel). GAD + -dentate neurons (e, f, black arrows) are readily identified. Degeneration and/or sprouting of residual Purkinje cell axons is evident (h, small black arrows and inset). There is artifactual staining of atrophic dentate neurons (h). Scale bars: 500 mum, A, B; 100 mum, C, D, and E, F (top panel), G, H. 50 mum, E, F (bottom panel), H, inset
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Fig3
GAD antibodies that labels predominantly GABA-ergic Purkinje cell afferents, including distinct puncta around large GAD-negative neurons (Fig. 3e; carets in lower panel), was markedly reduced in the patient's dentate (Fig. 3f), consistent with the marked loss of Purkinje cells; occasional perineuronal puncta were enlarged in size (Fig. 3f, lower panel, small arrowhead), suggesting synaptic degeneration. GAD+ GABA-ergic neurons in the dentate, which project to the inferior olive, were readily detected in the patient's dentate (Fig. 3e and f, arrows). As some of the GAD+ axons still seen in the patient's dentate may be from processes of neurons intrinsic to the dentate, immunostain to calbindinD28k was also performed, further demonstrating marked synaptic loss from Purkinje cells and only scattered degenerate and/or sprouting axonal profiles in the patient's dentate (Fig. 3g and h).
Degenerative changes in the patient's ION (b, d, f, h, j) compared with a control (a, c, e, g, i) with LH&E stain (a-d), calbindinD28k immunostain (e, f, i, j) and GAD immunostain (g, h). Marked patchy neuronal loss in the patient's principal olive (b, d, f, j), with myelin pallor and loss of calbindinD28k-labeled olivary efferents in the hilum (b, f). Residual neurons in the principal olive are often atrophic (small black arrows, d). Olivary neurons and GAD+ nucleo-olivary staining are preserved in the medial accessory olive (black arrow in a, b, e, f, g, h). GABA-ergic nucleo-olivary afferents show some mild patchy loss predominantly in dorsal and lateral regions of the principal olive (h). The dorsal arm in the principal olive shows a region with more numerous calbindinD28k-labeled neurons and preserved GAD staining (red arrow, f, h, j). Scale bars: 250 mum, A, B, E, F, G, H; 200 mum, top panel in C and D, I, J; 50 mum, bottom panel in C and D
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Fig4
In the dorsal arm of the principal olive in this patient, there was a region with somewhat greater preservation, seen in both calbindinD28k and GAD immunostains (Fig. 4f-h, red arrows).
PMC5769208_03
Degenerative changes in the patient's ION (b, d, f, h, j) compared with a control (a, c, e, g, i) with LH&E stain (a-d), calbindinD28k immunostain (e, f, i, j) and GAD immunostain (g, h). Marked patchy neuronal loss in the patient's principal olive (b, d, f, j), with myelin pallor and loss of calbindinD28k-labeled olivary efferents in the hilum (b, f). Residual neurons in the principal olive are often atrophic (small black arrows, d). Olivary neurons and GAD+ nucleo-olivary staining are preserved in the medial accessory olive (black arrow in a, b, e, f, g, h). GABA-ergic nucleo-olivary afferents show some mild patchy loss predominantly in dorsal and lateral regions of the principal olive (h). The dorsal arm in the principal olive shows a region with more numerous calbindinD28k-labeled neurons and preserved GAD staining (red arrow, f, h, j). Scale bars: 250 mum, A, B, E, F, G, H; 200 mum, top panel in C and D, I, J; 50 mum, bottom panel in C and D
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Fig4
In the dorsal arm of the principal olive in this patient, there was a region with somewhat greater preservation, seen in both calbindinD28k and GAD immunostains (Fig. 4f-h, red arrows).
Degenerative changes in the patient's left (a, c) and right (b, d) ION at a more rostral level, with immunostains to calbindinD28k (a, b) and GAD (c, d). A distinct region with more numerous calbindinD28k-labeled neurons is again seen in the dorsal arm of the principal olive bilaterally along with preserved GAD staining (red arrows). There is patchy loss of GAD staining, more prominent in lateral and ventral regions of the principal olive in this region of ION. Scale bar: 250 mum, A-D
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Fig5
In an adjacent more rostral level of the medulla, this region of relative neuronal preservation was even more striking, and present bilaterally (Fig. 5a-d, red arrows). Loss of GAD+ GABA-ergic nucleo-olivary afferents was more prominent in this section, particularly involving lateral and ventral regions of the principal olive (Fig. 5c and d).