Histology of small intestine.

<p>H&E staining of (A) Control (B) SMA (C) SMA+PMO25 small intestine. Shortened and blunted villi (* asterisk) and intramural edema (^ arrow head) were present in the lamina propria layer in SMA mice, along with the distinct intestinal crypt architectural distortion (arrow). Quantification of the villus length (D) and crypt size (E) in mice. **P < 0.001, *P < 0.05. Scale bar = 100 μm.</p

Systemic delivery of PMO25 increased <i>SMN2</i> exon 7 inclusion and SMN protein expression in intestine.

<p>(A) Representative image of reverse transcriptional polymerase chain reaction (PCR) showed the partial increase of full-length <i>SMN2</i> in SMA mice after PMO25 treatment. (B) Quantitative real-time PCR of full-length <i>SMN2</i> to Δ7 <i>SMN2</i> transcript ratio. (C) Western blotting assay of human SMN protein in intestine tissues from SMA and PMO25 treated SMA mice. β–tubulin was used as loading control. (D) Semi-quantification of SMN protein relative to tubulin control. Data were normalized to the ratio of SMN/tubulin in untreated SMA mice. (N = 3, *P< 0.05)</p

Increased macrophage infiltration in the gut of SMA mouse.

<p>(A) Representative image of macrophage staining in duodenum segment in SMA, control and PMO25 treated mice. Macrophages were stained with F4-80 antibody (green) and nuclei were stained with DAPI (blue). The absolute macrophage numbers per area in duodenum (B) and ileum (C) in three groups of mice. (N = 6. * P < 0.05; ** P < 0.01). Scale bar = 25 μm.</p

Antibodies used in immunohistochemical analysis.

<p>Antibodies used in immunohistochemical analysis.</p

Gross anatomical features of the bowel in SMA, control and PMO25 treated SMA mice.

<p>(A) Images of the whole bowel from 10 day old SMA (N = 3), control (N = 3) and PMO25 treated SMA mice (N = 3). Specimens were pinned in order to display the whole bowel length. Arrows indicate proximal duodenum and arrowheads identify the cecum. (B) Representative image of SMA and PMO25 treated SMA mice at PND10. (C) SMA mice had the shortest whole bowel length compared to control and PMO25 treated mice. (P< 0.0001 vs control and vs SMA+PMO25. N = 6–7). (D) The mean small bowel length was significantly lower in SMA mice than in control and treated mice (P< 0.0001 vs control and vs SMA+PMO25 in small bowel. N = 3–4). (E) SMA mice displayed the lowest body weight compared to control and PMO25 treated mice. (P< 0.0001 vs control and P<0.0001 vs SMA+PMO25. N = 3–4). The relative total intestine length to body weight (F) and relative small intestinal length to body weight (<b>G</b>) in SMA mice were significantly higher than those in control and PMO25 treated mice (P<0.0001 N = 3–4). ***P < 0.001, *P<0.05.</p

Blood vessel density in duodenum and ileum.

<p>(A) Representative image of vWF Immunofluorescence staining in duodenum and ileum of small intestine in control, SMA and PMO25 treated SMA mice. Blood vessels were indicated by vWF (red) staining. DAPI (blue) stains DNA nuclear and was used to outline the intestinal structure. Proportion of vascular density in duodenum (B) and ileum (C). The vascular density was quantified as pixels/unit area using imageJ software. Values in all three groups were then normalized to the mean value in the group of untreated SMA mice. Vascular density was significantly reduced in SMA mice in duodenum (P < 0.001 <i>vs</i> control, P <0.01 <i>vs</i> SMA+PMO25) and ileum (P < 0.05 <i>vs</i> control, P<0.05 <i>vs</i> SMA+PMO25), and was significantly improved after PMO25 treatment. (* P < 0.05, ** P<0.01). Scale bar = 50 μm.</p

Quantification of regeneration in BMD and DMD.

<p>(A) Regenerating myofibres (i.e. fetal and developmental myosin positive), quantified in 3 BMD (green) and 6 DMD (red) muscle biopsies show variable regeneration levels in BMD. Counts obtained from 10 random fields (except P7 and P9 where 9 fields from each were quantified) and expressed as a percentage of the total number of fibres. (B) Pooled analysis of total regenerating fibres showed double the number in DMD compared to BMD and (C) increased mean intensity of both myosins was seen in DMD ((n = 100), developmental; (n = 124), fetal total fibres) compared to BMD ((n = 18), developmental; (n = 27), fetal total fibres analysed) muscle fibres when sections were stained with either fetal or developmental myosin only. No regenerating fibres were present in BMD muscle section P5. Kruskal-Wallis followed by Dunn’s test performed (median and IQR depicted; * p<0.05; ** <i>p</i><0.01; *** <i>p</i><0.001, n/s—not significant).</p

Correlation of the percentage of regenerating fibres with functional motor score in BMD and DMD.

<p>Correlation between functional motor score (assessment from a total score of 40) and the level of regeneration in quadriceps muscle from BMD (n = 2) and DMD (n = 6) patients. Dashed lines show 95% confidence intervals.</p

Normalised utrophin intensity (mean ± standard error of mean).

<p>Normalised utrophin intensity (mean ± standard error of mean).</p

Muscle biopsies used in study.

<p>Muscle biopsies used in study.</p