Table 1.
Sex, age, anatomic localization of the lesion, morphologic diagnosis, distribution of the lesion, severity of the lesion and expression of p75NTR in dogs suffering non-suppurative meningoencephalitis of unknown origin, degenerative neuropathy and control dogs.
Fig 1.
Double immunofluorescence staining in a control sciatic nerve of a 4 week-old dog (A-E) and sciatic nerve of a dog with degenerative neuropathy (F-J).
In the control sciatic nerve, there is co-localization of p75NTR (red) with GAP-43 (green, arrows, A), GFAP (green, arrows, B), and nuclear Sox2 (green, arrows, C), thus most probably representing immature and/or non-myelinating Schwann cells. Co-expression of p75NTR (red) and Egr2/Krox20 (green, arrows, D) suggests a transition state between an immature and a myelinating Schwann cell phenotype. Periaxin expression (green, E) does not co-localize with p75NTR (red) in any cell, substantiating that p75NTR is completely down-regulated, once Schwann cells begin to myelinate. In contrast to the healthy nerve, there is no co-expression of p75NTR (red) with GAP-43 (green, F) and GFAP (green, G) in the case of degenerative neuropathy. However, co-localization of p75NTR with nuclear Sox2 (green, arrows, H) is evident, suggesting a dedifferentiated Schwann cell phenotype. Egr2/Krox20 (green, I) and periaxin (green, J) do not show co-labeling with p75NTR (red) in any cell. Nuclear counterstaining (blue) with bisbenzimide. (A), (B), (D), (F), (G), (I) and (J) scale bars: 100 μm. (C), (E) and (H) scale bars: 50 μm.
Fig 2.
Double immunofluorescence staining in a representative case of canine non-suppurative meningoencephalitis of unknown etiology (dog number 18).
Co-expression of p75NTR (red, A) and Sox2 (green, A) is observed in bipolar cells (arrow), morphologically resembling Schwann cells, which are localized in close proximity to a blood vessel. Note that few p75NTR-expressing cells lack Sox2 expression (arrowhead). In contrast to Sox2, GAP-43 (green, B), GFAP (green, C), Egr2/Krox20 (green, D), periaxin (green, E), and PDGFR-α (green, arrowhead, F) are not co-expressed with p75NTR (red), thus implying that p75NTR/Sox2-positive cells might represent a dedifferentiated Schwann cell phenotype within the injured CNS. Nuclear counterstaining (blue) with bisbenzimide. (A), (B) and (C) scale bars: 100 μm. (D), (E) and (F) scale bars: 50 μm.
Fig 3.
The immunoreaction of the peripheral myelin protein periaxin (brown, A) is limited to cranial nerves and abruptly stops upon entry of axons into the CNS in the brain stem of a control dog (dog number 27).
In non-suppurative meningoencephalitis of unknown origin (dog number 18, B), intralesional periaxin-positive structures (brown) appear as round to oval, small, ring-like foci with occasional clear central cores, most probably representing myelinating Schwann cells ensheathing an axon. Double immunohistochemistry for periaxin (brown, C) and p-NF (phosphorylated neurofilament, green, C) verifies that a large proportion of periaxin-positive Schwann cells (brown) in fact enwrap axons (green), substantiating effective Schwann cell remyelination. (A) scale bar: 50 μm. (B) and (C) scale bars: 100 μm. Double immunofluorescence staining for periaxin (green, D) and P0 (red, E) in a representative case of canine non-suppurative meningoencephalitis of unknown etiology (dog number 18) reveals that virtually all periaxin-positive Schwann cells co-express P0 (F). Nuclear counterstaining (blue) with bisbenzimide. (D), (E) and (F) scale bars: 100 μm. Double immunofluorescence staining for p75NTR (red) and periaxin (green) in the white matter (dog number 18; G) and brain stem (dog number 20; H) of representative cases of canine non-suppurative meningoencephalitis demonstrates increased Schwann cell remyelination depicted by periaxin immunoreactivity (green) in the brain stem compared to the cerebral white matter. Note that there is no co-localization of both markers. Nuclear counterstaining (blue) with bisbenzimide. (G) and (H) scale bars: 100 μm.
Fig 4.
Representative immunohistochemical staining of the brain stem and box-plots of cerebral and cerebellar white matter and brain stem illustrating differences in the number of p75NTR-(A–F), PDGFR-α-(G–L) and β-APP-(M–R) positive cells per mm² in p75NTR-/PRX-, p75NTR+/PRX-, and p75NTR+/PRX+ areas as well as unlesioned control tissue.
The number of p75 neurotrophin receptor (p75NTR) immunopositive cells (A, B, C, D; brain stem; brown) in the cerebral and cerebellar white matter (E) and brain stem (F) is significantly higher in p75NTR+/PRX+ areas in comparison to p75NTR+/PRX- areas. There are no differences in the number of PDGFR-α-positive OPCs (G, H, I, J; brain stem; brown; arrows) between any groups of lesioned areas and control tissue, localized in the cerebral and cerebellar white matter (K). In the brain stem (L), p75NTR+/PRX+ areas display significantly higher numbers of PDGFR-α-positive OPCs compared to p75NTR-/PRX-, p75NTR+/PRX- areas and control tissue. The number of β-APP-positive axons (M, N, O, P; brain stem; brown; arrows) in all lesioned areas within the cerebral and cerebellar white matter (Q) does not show significant differences between the lesioned areas and controls. In brain stem (R) p75NTR-/PRX- areas show a significantly higher number of β-APP-positive axons compared to p75NTR+/PRX-, p75NTR+/PRX+, and control tissue. Significance was designated as p < 0.05, as detected by Mann-Whitney-U group wise test. All box-plots show median value, lower and upper quartiles, minimum and maximum (excluding outliers). Black circle: extreme value; grey circle: outlier; asterisk: significant differences between respective groups. Scale bars: (A–D) and (M) 100 μm; (G–J), (N) and (O) 50 μm; (P) 20 μm.
Fig 5.
Representative immunohistochemical and lectin histochemical staining of the brain stem and box-plots of cerebral and cerebellar white matter and brain stem illustrating differences in the number of GFAP-(A–F), CD3-(G–L) and BS-1-(M–R) positive cells per mm² in p75NTR-/PRX-, p75NTR+/PRX-, and p75NTR+/PRX+ areas as well as unlesioned control tissue.
GFAP-positive cells (A, B, C, D; brains stem; brown) are significantly higher in number in all lesioned groups of the white matter (E) compared to control tissue. There is no difference in GFAP immunoreactivity in the brain stem compared to controls (F). CD3-positive cells (G, H, I, J; brain stem; brown) are significantly lower in number in p75NTR+/PRX+ and p75NTR+/PRX- lesions, when compared to p75NTR-/PRX- areas in the cerebral and cerebellar white matter (K) and in the brain stem (L). The number of BS-1-positive microglia/macrophages (M, N, O, P; brain stem; brown) lacks significant variations between the lesioned groups in any anatomical localization (Q, R). Significance was designated as p < 0.05, as detected by Mann-Whitney-U group wise test. All box-plots show median value, lower and upper quartiles, minimum and maximum (excluding outliers). Black circle: extreme value; grey circle: outlier; asterisk: significant differences between respective groups. Scale bars: (A–D) 100 μm; (G–J) and (M–P) 50 μm.
Fig 6.
Double immunofluorescence staining of p75NTR (red) and Sox2 (green), p75NTR and nuclear counterstaining with bisbenzimide (blue), and merged pictures of p75NTR, Sox2, bisbenzimide at day 9 of cultivation (A,B,C) and day 18 of cultivation (D,E,F) in organotypic slice cultures from the normal adult canine brain stem.
During culturing, relatively small numbers of p75NTR (red) and Sox2 (green, nuclear staining) co-expressing cells (arrows) are detected in the brain parenchyma in close proximity to blood vessels by day 9 (A,B,C), but increase in number by day 18 (D,E,F) of culturing. (A-F) scale bars: 20 μm. BS-1 lectin histochemistry (G-J) demonstrates the lack of microglial and macrophage-like cells at the beginning of the cultivation period (G; 0 days) and a massive increase in the number of BS1-positive macrophages/microglia during cultivation (H, 3 days; I, 9 days), reaching their highest numbers by day 18 of culturing (J). (G-J) scale bars: 50 μm.