Spread of OnabotulinumtoxinA After Bladder Injection Spread of OnabotulinumtoxinA After Bladder Injection. Experimental Study Using the Distribution of Cleaved SNAP-25 as the Marker of the Toxin Action  Ana Coelho, Francisco Cruz, Célia D. Cruz, António Avelino  European Urology  Volume 61, Issue 6, Pages 1178-1184 (June 2012) DOI: 10.1016/j.eururo.2012.01.046 Copyright © 2012 European Association of Urology Terms and Conditions

Fig. 1 Guinea pig bladder diagrams showing the distribution of cleaved synaptosome-associated protein of 25 kDa immunoreactive fibers (dark lines). A total of 2 U of onabotulinumtoxinA (Onabot/A) diluted in 2μl (upper diagrams) or 20μl (lower diagrams) of saline were administered in the indicated area (syringes). Transverse sections taken at the level of the ureters were analyzed. European Urology 2012 61, 1178-1184DOI: (10.1016/j.eururo.2012.01.046) Copyright © 2012 European Association of Urology Terms and Conditions

Fig. 2 Average number of immunoreactive fibers for cleaved synaptosome-associated protein of 25 kDa (SNAP-25) observed per section after onabotulinumtoxinA (Onabot/A) intramural injection diluted in 2μl or 20μl of saline. Six bladder sections were analyzed per animal. The difference has statistical significance. * p<0.05; error bars show standard deviation. European Urology 2012 61, 1178-1184DOI: (10.1016/j.eururo.2012.01.046) Copyright © 2012 European Association of Urology Terms and Conditions

Fig. 3 Expression of cleaved synaptosome-associated protein of 25 kDa (cSNAP-25) using the avidin-biotin method after onabotulinumtoxinA administration (A) through intravesical instillation or (B, C) through an intramural injection. Dashed lines indicate the boundary between the urothelium and lamina propria. (a) Animals that received intravesical instillation of toxin did not express cSNAP-25 either in the urothelium and mucosa or in the muscular layer. (B) In animals that received an intramural injection of toxin, immunoreactive fibers are observed in the suburothelium present in the lamina propria and in fibers penetrating the urothelium. In (C) the muscular layer, immunoreactive fibers run parallel to the detrusor smooth muscle (S). Magnification bars: 50μm. European Urology 2012 61, 1178-1184DOI: (10.1016/j.eururo.2012.01.046) Copyright © 2012 European Association of Urology Terms and Conditions

Fig. 4 Coexpression of cleaved synaptosome-associated protein of 25 kDa (cSNAP-25) (red) with vesicular acetylcholine transporter (VAChT) (A, green), tyrosine hydroxylase (TH) (B, green), and calcitonin gene-related peptide (CGRP) (C, green) after onabotulinumtoxinA (Onabot/A) administration throughout the whole bladder. Note the extensive colocalization found between cSNAP-25 and VAChT (A, yellow/orange); (D) quantification of the coexpression. *** p<0.0001. Magnification bars: 50μm. European Urology 2012 61, 1178-1184DOI: (10.1016/j.eururo.2012.01.046) Copyright © 2012 European Association of Urology Terms and Conditions

Fig. 5 Percentage of (A) parasympathetic, (B) sympathetic, or (C) sensory fibers that also express cleaved synaptosome-associated protein of 25 kDa (cSNAP-25) at different time points after onabotulinumtoxinA (Onabot/A) intramural injection throughout the whole bladder. After 24h, 3 d, or 7 d after (Onabot/A) injection, the number of cSNAP-25 and the percentage of colocalization is unchanged. CGRP=calcitonin gene-related peptide; TH=tyrosine hydroxylase; VAChT=vesicular acetylcholine transporter. European Urology 2012 61, 1178-1184DOI: (10.1016/j.eururo.2012.01.046) Copyright © 2012 European Association of Urology Terms and Conditions