However, a significant reduction in IL-17 was observed in the spleen of EAE anatabine treated mice compared to EAE placebo mice (Fig. 2C)
This particular dosage was selected from a previous study showing that this dosage is efficient at lowering brain cytokine levels in a mouse model of Alzheimer’s disease displaying chronic neuroinflammation
Clinical signs of EAE became apparent by day 6 following the immunization in the placebo group but were delayed to day 11 in the anatabine treatment group (Fig. 1A)
The average clinical severity of EAE was also significantly reduced by the anatabine treatment (Fig. 1B)
Importantly, approximately 70% of the mice in the placebo group developed hind-limb weakness or paralysis compared to only 20% in the anatabine treatment group (Fig. 1C) showing that the mice treated with anatabine displayed significantly milder disease symptoms than the placebo group
However, anatabine significantly suppressed the production of IL-1b, IL-6 and IL-17A in the serum of EAE mice (Fig. 2B) but did not significantly impact IFN-gamma and TNF-alpha
Anatabine appears to fully suppress IL-1b, INF-gamma and TNF-a elevation in the spleen of EAE mice (Fig. 2C).
We have shown previously that anatabine prevents STAT3 and p65 NFkB phosphorylation in various cell types and suggested that anatabine impact cytokine production by this mechanism as both STAT3 and p65 NFkB are known to regulate cytokine production.
Interestingly, a significant increase in STAT3 phosphorylation was observed in the spleen of EAE mice compared to control non immunized mice and a significant decrease in STAT3 phosphorylation level was detected in EAE mice treated with anatabine (Fig. 3)
Additionally, we observed that anatabine significantly prevented STAT3 and p65 NFkB phosphorylation in brain homogenates of EAE mice (Fig. 4)
A reduction in the number of phosphorylated STAT3 immunoreactive cells was observed in the cerebral cortex of EAE mice treated with anatabine further confirming the data obtained by western-blotting using brain homogenates (Fig. 5)
Similarly, we observed an increased number of phosphorylated STAT3 immunopositive cells in the spinal cord of EAE mice compared to control non-immunized mice and a decreased number of phosphorylated STAT3 immunoreactive cells in EAE mice treated with anatabine (Fig. 6) showing overall that anatabine reduces STAT3 phosphorylation in the brain, spinal cord and spleen of EAE mice.
Overall, anatabine did not significantly impact EAE induced astrogliosis for the different areas of the brain examined (Fig. 7)
A marked increase in GFAP immunostaining was also observed in the spinal cord of EAE mice and was significantly suppressed by the anatabine treatment showing that anatabine prevents astrogliosis in the spinal cord of EAE mice (Fig. 11)
Interestingly, a significant reduction in the number of IBa1 immunopositive microglial cells was found in the hippocampus and medulla of anatabine treated EAE mice (Fig. 8)
Less cellular infiltration was observed in the spinal cord of anatabine treated mice compared to EAE placebo mice (Fig. 9)
In addition, anatabine markedly decreased the Iba1 immunostaining in the spinal cord of EAE mice showing that anatabine reduces the infiltration of macrophage/ microglia in the spinal cord of EAE mice (Fig. 10)
Interestingly, anatabine significantly prevented demyelination associated with EAE in the spinal cord (Fig. 12)
A statistically significant positive correlation was observed between the amount of GFAP and Iba1 burden in the spinal cord (Pearson correlation = 0.612, P,0.002) whereas negative correlations were observed between the GFAP burden and the Luxol fast blue burden (Pearson correlation =20.525, P= 0.007), and between the IBa1 burden and the Luxol fast blue burden (Pearson correlation =20.609, P =0.001) suggesting that astrogliosis and microgliosis are associated with the loss of myelin in the spinal cord
The axonal damage in EAE mice leads to well defined clinical signs such as tail paralysis, hind-limb weakness and paralysis
We found that IL-1b, IFN-gamma, TNF-alpha, IL-6 and IL-17A were significantly elevated in the serum of EAE mice compared to control non-immunized mice
In addition, we observed a statistically significant correlation between the amount of p65 NFkB phosphorylation and STAT3 phosphorylation in brain homogenates (Pearson Correlation = 0.637, P<0.001)
Interestingly, a statistically significant correlation was observed between the amount of STAT3 phosphorylation detected in brain homogenates and the clinical severity of EAE (Pearson correlation = 0.653, P<0.001) but not with p65 NFkB phosphorylation (Pearson correlation = 0.371, P= 0.062) suggesting that STAT3 may play a more preponderant role than NFkB in the development of neurological deficits of EAE
A statistically significant correlation was observed between the number of STAT3 phosphorylated cells in the cerebral cortex and the clinical severity of EAE (Pearson Correlation =0.531, P,0.002)
A statistically significant correlation was also observed between the amount of STAT3 phosphorylated cells in the spinal cord and the clinical severity of EAE (Pearson Correlation = 0.512, P,0.004)
A significant elevation of IL-1b, INF-gamma and TNF-alpha was observed in the spleen of EAE mice compared to control non immunized animals
Western-blot experiments using brain homogenates from EAE mice also reveal that both STAT3 and p65 NFkB phosphorylation are significantly elevated in the brain of EAE mice compared to control non immunized mice (Fig. 4)
We further assessed STAT3 phosphorylation by immunohistochemistry using brain sections of the animals and observed an increased number of phosphorylated STAT3 immunopositive cells in the cortex of EAE mice compared to control non-immunized mice (Fig. 5)
A significant increase in astrogliosis revealed by a GFAP immunostaining was observed in the cortex and medulla of EAE mice compared to control non immunized animals (Fig. 7)
A significant increase in the number of microglial cells immunoreactive for Iba1 was observed in the hippocampus and medulla of EAE mice compared to control non-immunized mice (Fig. 8)
As shown in Fig. 9, hematoxylin and eosin staining revealed numerous perivascular clusters of mononuclear infiltrating cells in the spinal cord of EAE mice and an absence of these inflammatory cell infiltrates in the spinal cord of control non-immunized mice
Globally, Iba1 immunostaining is significantly increased in the spinal cord of EAE mice compared to control nonimmunized mice (Fig. 10)
Finally, Luxol Fast Blue staining showed significant myelin loss in the white matter of the spinal cord of EAE mice compared control non-immunized mice (Fig. 12)
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If you find BEL Commons useful in your work, please consider citing: Hoyt, C. T., Domingo-Fernández, D., & Hofmann-Apitius, M. (2018). BEL Commons: an environment for exploration and analysis of networks encoded in Biological Expression Language. Database, 2018(3), 1–11.