Fig 1.
Immune cell infiltration and heterokaryon formation in EAE mouse cerebella.
(A) Comparison between coronal sections of cerebella from Control (left) and EAE immunized mice (right). There is a higher number of GFP-labeled infiltrating bone marrow-derived cells and Purkinje heterokaryons (arrows) in EAE affected mice as compared to control animals. Scale bar 300 μm. (B) Quantification of Purkinje heterokaryons in Control (n = 3; 1.0 ± 0.6) and EAE (n = 7; 77.7 ± 16.7) shows a significant difference (p = 0.0167, Mann-Whitney-Wilcoxon test). (C) In EAE, more heterokaryons were located in the vermis (54.4 ± 12.0) than in the lateral hemispheres (n = 7; 23.3 ± 5.0) (n = 7, p = 0.0313).
Fig 2.
Immune cell infiltration in the spinal cord of Control and EAE affected mice.
Coronal spinal cord sections showed little infiltration of GFP-labeled bone marrow-derived cells (green) in control (left) animals, while infiltration in EAE immunized animals (right) was prominent. Scale bar 300 μm.
Fig 3.
Formation of a heterokaryon in the spinal cord.
(A) A motor neuron that is present in the ventral horn of the spinal cord expresses GFP (arrow) as a result of fusion between a GFP expressing bone marrow-derived cell and a motor neuron. The arrow shows a single GFP-labeled motor neuron in the ventral horn of the spinal cord. Scale bar 300 μm. (B) Higher magnification of a spinal cord GFP-labeled motor neuron shown in (A). Scale bar 150 μm. (C-E) Z-stack images of the GFP-labeled motor neuron shown in (A) and (B). (D-E) Immunohistochemistry demonstrating that the GFP-labeled motor neuron co-expresses GFP and NeuN. (F) Triple staining of the same motor neuron, NeuN (red), GFP (green) and Hoechst (blue). Two nuclei (Hoechst, blue) are present in the same cell, marked with dotted circles thus it is a heterokaryon. Scale bar 25 μm.
Fig 4.
GFP-labeled spinal cord motor neuron co-expressing NeuN.
(A) GFP-labeled (green) ventral horn motor neuron (arrow) extending a single axon from the grey matter (GM) to the white matter (WM) (see arrowheads). (B-C) This GFP-labeled motor neuron co-expresses NeuN. (D-F) Higher magnification of the motor neuron in B-C. Scale bar (A-C) 150 μm, (D-F) 25 μm.
Fig 5.
Quantification and distribution of heterokaryons in EAE affected and Control spinal cord.
(A) While there was a wide spread in the number of heterokaryons between individual EAE affected mice (n = 5; 20.0 ± 6.7), depending on the severity of inflammation, there were significantly more heterokaryons (p = 0.0358, Mann-Whitney-Wilcoxon test) in EAE affected mice than Control (n = 3; 0.3 ± 0.3) mice. (B) Schematic representation of the distribution of heterokaryons in 20 sections of EAE spinal cord. Each symbol represents one experimental animal, and the symbol size represents heterokaryon size (small symbol: <20 μm, large: >20 μm).
Fig 6.
GFP-labeled motor neurons and interneurons in the spinal cord.
(A) In a coronal section of the spinal cord with prominent infiltration of GFP-labeled bone marrow-derived cells (green), a number of GFP-labeled interneurons were detected. (B) Magnification of the area indicated with a box in (A). Eight GFP-labeled interneurons (green, indicated by arrows) located in the grey matter (GM), and one motor neuron (arrowhead) extending an axon/dendrites across the white matter (WM). (C) Most of these cells co-label with NeuN (indicated by arrowheads). (D) Schematic illustration of GFP-labeled interneuron and their dendrites. Scale bar (A-D) 150 μm.