Histopathological Defects in Intestine in Severe Spinal Muscular Atrophy Mice Are Improved by Systemic Antisense Oligonucleotide Treatment

Acknowledgments This study is supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London (FM and HZ), the Medical Research Council grant (grant reference MR/L013142/1, FM), SMA-Europe grant (FM and HZ) and Great Ormond Street Hospital Children’s Charity grants (FM and HZ). JEM is supported by Great Ormond Street Hospital Children’s Charity. PS is supported by Bill Marshall Fellowship and The CP Charitable Trust at Great Ormond Street Hospital and UCL. SHP is supported by SMA Trust and Euan MacDonald Centre for Motor Neurone Disease Research.Peer reviewedPublisher PD

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

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

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

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

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