Combination therapy with EGCG and GA delays disease onset and synergistically reduces EAE severity.

<p>For preventive treatment EGCG (300 µg) or vehicle (NaCl 0.9%) was administered orally twice daily from day −9 before immunization with PLP_139–151 until day 45. GA (50 µg) or vehicle (Mannitol 4%) in incomplete Freund's adjuvant was given s.c. once on day −7 before immunization. Mean disease score of control, EGCG, GA and the combination therapy group is shown from day 0 to day 45 after immunization (A), * p<0.05 day 13,15–18,20–22,24,26–27, **p<0.01 day 14,19,23,25,28,35–39,42, n = 8 per group. Onset of disease in control, GA, EGCG and combination therapy group is presented in a Kaplan-Meier survival curve as percentage of sick mice at a certain day after immunization (B), *p<0.05. Cumulative disease activity of control, GA, EGCG and combination therapy group was calculated as the area under the curve of the clinical score plots for each individual animal (C), *p<0.05.</p

Combination therapy with EGCG and GA alleviates disability in established EAE.

<p>For treatment of acute disease PLP_139–151-induced EAE mice were randomized into two groups after reaching a disease score of 2 and subsequently received 300 µg of oral EGCG or vehicle (0.9% NaCl) twice daily in addition to daily s.c. injections of 150 µg GA or vehicle (4% Mannitol). Mean disease score of control and combination therapy group is shown from day 0 to day 44 after immunization (A), *p<0.05 day 19, 20, 23, 24, 34, n = 6. Cumulative disease activity of control and combination therapy group was calculated (B), *p<0.05.</p

EGCG and GA together ameliorate CNS inflammatory pathology.

<p>Representative Hematoxylin and Eosin staining of transverse spinal cord sections of vehicle, EGCG, GA and EGCG and GA treated EAE mice is shown (A). 100× magnification, scale bar 500 µm. Inflammation in standardized areas of the spinal cord of all treatment groups was assessed and is presented semi-quantitatively as percentage of spinal cord quadrants with inflammatory foci relative to all assessed quadrants (B), *p<0.05, n = 8 per group.</p

EGCG and GA promote axonal growth and density <i>in vitro</i>.

<p>Representative photomicrographs of brain slices with axonal outgrowth after addition of EGCG (5 µg/ml), GA (6.25 µg/ml) and the combination of EGCG (5 µg/ml) and GA (6.25 µg/ml) are shown. 100× magnification, scale bar: 300 µm (A). The number of axons was measured by quantifying all intersections of axons with a standardized line parallel to the brain slice border, **p<0.001 (B). The density of outgrowing axons was determined by measuring the mean intensity in a standardized area parallel to the brain slice edge, **p<0.001 (C). Levels of GDNF (D) and BDNF (E) were measured in the supernatant of outgrowth assays by ELISA, n.d. = not detectable. **p<0.001 ***p<0.0001.</p

EGCG and GA in combination inhibit death of CNS cells <i>in vitro</i>.

<p>Survival of murine HT22 neuronal cells (A) and human glioblastomal LN18 cells (B) was determined in a crystal violet assay after induction of apoptosis by incubation with Glutamate (5 mM) (A) or TRAIL (20 ng/ml) (B). EGCG (10 µg/ml) and/or GA (12.5 µg/ml) were added to the culture 2 h before apoptosis induction, *p<0.05, **p<0.001, representative of 2 experiments.</p