Factor VIII-related antigen

In control dogs, factor VIII-related antigen expression was detected in endothelial cells and to a lesser extent in the subendothelial matrix of the vessel wall. The expression was mainly restricted to the wall of meningeal and parenchymal blood vessels as a medium to dark brown signal.

In the cerebellum of CDV-infected dogs, the same reaction pattern was observed (fig.

27 A-D). Additionally, in subacute and chronic lesions, an overall increase in factor VIII-related antigen expression was noted. Conversely, some chronic lesions showed a decrease of the signal in the center of the lesion.

Quantitatively, the median of the factor VIII-positive area related to the total lesioned area in group 1, 2 and 3 was of 1.23%, 0.84% and 0.92%, respectively. A median value of 1.16% for group 4 and 1.06% for group 5 was detected. There was a slight increase to a median of 2.03% in group 6. In addition, the quantitative evaluation of factor VIII for group 7 revealed a median of 1.79%. The median in group 8 lesions was of 2.27% with a maximum of 3.80 % in group 8 (fig. 28). The original data (median, minimum and maximum) can be found in chapter 9.4, table 9.4.2.

A B

C D

Figure 27 A: Cerebellum, animal no. 16, white matter, control dog, strong labeling of Factor VIII around basement membranes. B: Cerebellum, animal no. 8, white matter, acute lesion, mild to moderate labeling of Factor VIII in BMs of blood vessels. C: Cerebellum, animal no. 14, white matter, subacute lesion with inflammation, mild to moderate expression of Factor VIII in basement membranes. D: Cerebellum, animal no. 14, white matter, chronic lesion, a mild to moderate decrease of Factor VIII labeling in the center of the lesion; Factor VIII immunohistochemistry (DAB), scale bar = 50 µm.

Figure 28: Morphometrcally identified factor VIII-related antigen expression related to the investigated white matter area or total lesion area in controls and DL animals

1 = control, 2 = normal appearing white matter (NAWM), 3 = antigen detection without obvious lesion, 4 = vacuolation, 5 = acute, 6 = subacute without inflammation, 7 = subacute with inflammation, 8 = chronic. Columns display values as median, minimum, maximum and the lower and upper quartile. Small circle: extreme value;

small asterisk: outlier.

In the statistical analysis using a one-way ANOVA followed by multiple pair-wise comparisons of geometric means with alpha-adjustment by Tukey-Kramer test, a significant difference was found between the groups (p <0.05). Group 6 and 8 showed a statistically significant difference in Factor VIII expression compared to group 2, 3 and 4. Group 7 revealed significant differences to group 2. In addition the

group 5 differed significantly from group 2 and 3. The p values can be found in chapter 9.4, table 9.4.3.

Figure 29: Geometric mean and geometric standard deviation of factor VIII-related antigen expression in controls and DL animals

0 0,5 1 1,5 2 2,5 3 3,5 4

1 2 3 4 5 6 7 8

Groups Factor VIII-positive area (% of investigated white matter area or total lesion area)

1 = control, 2 = normal appearing white matter (NAWM), 3 = antigen detection without obvious lesion, 4 = vacuolation, 5 = acute, 6 = subacute without inflammation, 7 = subacute with inflammation, 8 = chronic. Factor VIII = factor VIII-related antigen. Columns display values as geometric mean and geometric standard deviations.

The geometric mean and geometric standard deviation of factor VIII expression for each group can be found in chapter 9.4, table 9.4.4.

4.4.4 Aggrecan

In the cerebellum of control dogs, aggrecan immunoreactivity was observed in the molecular and granular layers as extracellular finely dispersed, brown granules with a marked perineuronal pattern around the Purkinje cells. This granular reaction extended into the white matter and surrounded neuronal cell bodies and their dendrites. In addition, a mild positive reaction was observed around and in glial cells (fig 30 A).

The same reaction pattern was found in group 2 (NAWM) areas, whereas in group 3 to 7 an additional intralesional extracellular aggregation of fine brown granules was detected (fig. 30 B, C). In the center of chronic plaque (group 8), there was an overall decrease in aggrecan expression, however an accumulation of aggrecan-positive material in foamy macrophages at the edge of the lesions was also detected (fig. 30 D).

Quantitatively, the median value of the positive area related to the total lesion area for group 1 was 0.01%, 0.04% for group 2 and 0.07% for group 3. Group 4 showed a median value of 0.13%, 0.08% for group 5. In subacute lesions without inflammation (group 6), the median value was 0.12%. The quantitative value was 0.11% in group 7 and there was an increase of aggrecan expression in group 8 up to 0.18% (fig. 31).

The original data (median, minimum and maximum) can be found in chapter 9.4, table 9.4.2.

A B

C D

Figure 30 A: Cerebellum, animal no. 16, white matter, control dog, mild extracellular expression of aggrecan. B: Cerebellum, animal no. 8, white matter, acute lesion, mild expression of aggrecan in glia cells C: Cerebellum, animal no. 14, white matter, subacute lesion with inflammation, mild expression of aggrecan in glia cells. D: Cerebellum, animal no. 14, white matter, chronic lesion, prominent focal intralesional aggrecan deposition mainly in macrophages (arrows). Aggrecan immunohistochemistry (DAB); scale bar = 50 µm.

Figure 31: Morphometrically identified proportion of aggrecan-positive area related to the investigated white matter area or total lesion area

1 = control, 2 = normal appearing white matter (NAWM), 3 = antigen detection without obvious lesion, 4 = vacuolation, 5 = acute, 6 = subacute without inflammation, 7 = subacute with inflammation, 8 = chronic. Columns display values as median, minimum, maximum and the lower and upper quartile. Small circle: extreme value;

small asterisk: outlier

Statistical analysis using a one-way ANOVA followed by multiple pair-wise comparisons of geometric means (fig. 32) with alpha-adjustment by Tukey-Kramer test revealed a significant difference between the groups (p <0.05). Group 1 showed a highly statistically significant difference of aggrecan expression (p <0.0001) compared to group 2 to 8. In addition, group 2 revealed a significant difference compared to group 3 to 8. Group 3 also revealed a significant difference compared to group 4, 6, and 7. The p values can be found in chapter 9.4, table 9.4.3.

Figure 32: Geometric mean and geometric standard deviation of aggrecan-positive area related to the investigated white matter area or total lesion area

0 0,05 0,1 0,15 0,2 0,25 0,3 0,35 0,4

1 2 3 4 5 6 7 8

Groups Aggrecan-positive area (% of investigated white matter area or total lesion area)

1 = control, 2 = normal appearing white matter (NAWM), 3 = antigen detection without obvious lesion, 4 = vacuolation, 5 = acute, 6 = subacute without inflammation, 7 = subacute with inflammation, 8 = chronic. Columns display values as geometric mean and geometric standard deviations.

The geometric mean and geometric standard deviation of aggrecan expression for each group can be found in chapter 9.4, table 9.4.4.