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

Sintusek P, Catapano F, Angkathunkayul N, Marrosu E, Parson SH, Morgan JE, Muntoni F, Zhou H - PLoS ONE (2016)

Bottom Line: We also assessed the therapeutic response of GI histopathology to systemic administration of morpholino antisense oligonucleotide (AON) designed to increase SMN protein expression.Significant anatomical and histopathological abnormalities, with striking reduction of vascular density, overabundance of enteric neurons and increased macrophage infiltration, were detected in the small intestine in SMA mice.We conclude that the observed GI histopathological phenotypes and functional defects observed in these SMA mice are strongly linked to SMN deficiency which can be rescued by systemic administration of AON.

View Article: PubMed Central - PubMed

Affiliation: Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, United Kingdom.

ABSTRACT
Gastrointestinal (GI) defects, including gastroesophageal reflux, constipation and delayed gastric emptying, are common in patients with spinal muscular atrophy (SMA). Similar GI dysmotility has been identified in mouse models with survival of motor neuron (SMN) protein deficiency. We previously described vascular defects in skeletal muscle and spinal cord of SMA mice and we hypothesized that similar defects could be involved in the GI pathology observed in these mice. We therefore investigated the gross anatomical structure, enteric vasculature and neurons in the small intestine in a severe mouse model of SMA. We also assessed the therapeutic response of GI histopathology to systemic administration of morpholino antisense oligonucleotide (AON) designed to increase SMN protein expression. Significant anatomical and histopathological abnormalities, with striking reduction of vascular density, overabundance of enteric neurons and increased macrophage infiltration, were detected in the small intestine in SMA mice. After systemic AON treatment in neonatal mice, all the abnormalities observed were significantly restored to near-normal levels. We conclude that the observed GI histopathological phenotypes and functional defects observed in these SMA mice are strongly linked to SMN deficiency which can be rescued by systemic administration of AON. This study on the histopathological changes in the gastrointestinal system in severe SMA mice provides further indication of the complex role that SMN plays in multiple tissues and suggests that at least in SMA mice restoration of SMN production in peripheral tissues is essential for optimal outcome.

No MeSH data available.


Related in: MedlinePlus

Enteric neurons in SMA mouse small intestine.(A) Representative image of enteric neurons/ganglions in Duodenum myenteric plexuses from control, SMA and PMO25 treated SMA mice. Enteric neurons were stained with neuronal marker PGP9.5 (red). The muscular layer was stained with α-smooth muscle actin (green). Cell nuclei were stained with DAPI (blue). (B) Relative ganglion density in 3 groups of mice. Pixels of PGP9.5 immunostaining per captured field was used to quantify the ganglion density using imageJ software and expressed as pixels per unit area. Ganglion density was significantly increased in SMA mice in both duodenum (P = 0.028 vs control, N = 4 per group) and ileum (P = 0.018 vs control, N = 4 per group) and significantly decreased after PMO25 treatment (P = 0.038 in duodenum; P = 0.019 in ileum; N = 4 per group). (C) The mean number of neurons was also significantly increased in both duodenum (P = 0.0045 vs control, P< 0.001 vs PMO25 treatment) and ileum (P = 0.04 vs control, P = 0.012 vs PMO25 treated SMA) in SMA mice and was reduced significantly by PMO25 treatment (N = 6–8, * P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar = 25 μm.
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pone.0155032.g004: Enteric neurons in SMA mouse small intestine.(A) Representative image of enteric neurons/ganglions in Duodenum myenteric plexuses from control, SMA and PMO25 treated SMA mice. Enteric neurons were stained with neuronal marker PGP9.5 (red). The muscular layer was stained with α-smooth muscle actin (green). Cell nuclei were stained with DAPI (blue). (B) Relative ganglion density in 3 groups of mice. Pixels of PGP9.5 immunostaining per captured field was used to quantify the ganglion density using imageJ software and expressed as pixels per unit area. Ganglion density was significantly increased in SMA mice in both duodenum (P = 0.028 vs control, N = 4 per group) and ileum (P = 0.018 vs control, N = 4 per group) and significantly decreased after PMO25 treatment (P = 0.038 in duodenum; P = 0.019 in ileum; N = 4 per group). (C) The mean number of neurons was also significantly increased in both duodenum (P = 0.0045 vs control, P< 0.001 vs PMO25 treatment) and ileum (P = 0.04 vs control, P = 0.012 vs PMO25 treated SMA) in SMA mice and was reduced significantly by PMO25 treatment (N = 6–8, * P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar = 25 μm.

Mentions: The autonomic nervous system acting through the enteric nervous system (ENS) is responsible for the regulation and control of all gastrointestinal functions. Disrupted ENS signaling in intestine, resulting in defective GI function, has been reported in the adult stage of two mild mouse models of Smn deficiency [33]. In this study, we examined enteric neurons in the severe Taiwanese SMA mouse model, which has a severe phenotype and short lifespan. Ganglion density and neuron numbers in the myenteric plexuses were identified and quantified by PGP9.5 staining. To identify the muscular layer of the intestine wall, α-smooth muscle actin staining was used to discriminate myenteric from submucosal plexuses. Unexpectedly, the area occupied by ganglia of the myenteric plexus was significantly increased in SMA mice with more closely-packed neurons compared to control mice (Fig 4). The number of neurons and ganglion density in the myenteric plexuses were significantly greater in SMA compared to control mice, in both duodenum (ganglion density: 256.7±51.6 vs 132.7±10.7, N = 4, P<0.05; neurons: 156.9±16.3 vs 93.8±8.3, N = 6–8, P<0.01) and ileum (ganglion density: 137.9±22.6 vs 63.8±16.0, N = 4, P<0.05; neurons: 89.4±10.9 vs 65.1±3.6, N = 6–8, P<0.05). The ganglion density and number of neurons were significantly reduced to near-normal levels after PMO25 treatment in both duodenum (ganglion density: 256.7±51.6 (SMA) vs 136.5±21.9 (SMA+PMO25), N = 4, P<0.05; neuron number: 156.9± 16.3 vs 65.4± 8.1, N = 8, P<0.001) and ileum (ganglion density: 137.9±22.6 and 71.8±11.0, N = 4, P<0.05; neuron number: 89.4±10.9 vs 59.0±5.4, N = 8, P<0.05). There was no significant difference between all parameters in control and SMA+PMO25 mice (Fig 4).


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

Sintusek P, Catapano F, Angkathunkayul N, Marrosu E, Parson SH, Morgan JE, Muntoni F, Zhou H - PLoS ONE (2016)

Enteric neurons in SMA mouse small intestine.(A) Representative image of enteric neurons/ganglions in Duodenum myenteric plexuses from control, SMA and PMO25 treated SMA mice. Enteric neurons were stained with neuronal marker PGP9.5 (red). The muscular layer was stained with α-smooth muscle actin (green). Cell nuclei were stained with DAPI (blue). (B) Relative ganglion density in 3 groups of mice. Pixels of PGP9.5 immunostaining per captured field was used to quantify the ganglion density using imageJ software and expressed as pixels per unit area. Ganglion density was significantly increased in SMA mice in both duodenum (P = 0.028 vs control, N = 4 per group) and ileum (P = 0.018 vs control, N = 4 per group) and significantly decreased after PMO25 treatment (P = 0.038 in duodenum; P = 0.019 in ileum; N = 4 per group). (C) The mean number of neurons was also significantly increased in both duodenum (P = 0.0045 vs control, P< 0.001 vs PMO25 treatment) and ileum (P = 0.04 vs control, P = 0.012 vs PMO25 treated SMA) in SMA mice and was reduced significantly by PMO25 treatment (N = 6–8, * P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar = 25 μm.
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pone.0155032.g004: Enteric neurons in SMA mouse small intestine.(A) Representative image of enteric neurons/ganglions in Duodenum myenteric plexuses from control, SMA and PMO25 treated SMA mice. Enteric neurons were stained with neuronal marker PGP9.5 (red). The muscular layer was stained with α-smooth muscle actin (green). Cell nuclei were stained with DAPI (blue). (B) Relative ganglion density in 3 groups of mice. Pixels of PGP9.5 immunostaining per captured field was used to quantify the ganglion density using imageJ software and expressed as pixels per unit area. Ganglion density was significantly increased in SMA mice in both duodenum (P = 0.028 vs control, N = 4 per group) and ileum (P = 0.018 vs control, N = 4 per group) and significantly decreased after PMO25 treatment (P = 0.038 in duodenum; P = 0.019 in ileum; N = 4 per group). (C) The mean number of neurons was also significantly increased in both duodenum (P = 0.0045 vs control, P< 0.001 vs PMO25 treatment) and ileum (P = 0.04 vs control, P = 0.012 vs PMO25 treated SMA) in SMA mice and was reduced significantly by PMO25 treatment (N = 6–8, * P < 0.05; ** P < 0.01; *** P < 0.001). Scale bar = 25 μm.
Mentions: The autonomic nervous system acting through the enteric nervous system (ENS) is responsible for the regulation and control of all gastrointestinal functions. Disrupted ENS signaling in intestine, resulting in defective GI function, has been reported in the adult stage of two mild mouse models of Smn deficiency [33]. In this study, we examined enteric neurons in the severe Taiwanese SMA mouse model, which has a severe phenotype and short lifespan. Ganglion density and neuron numbers in the myenteric plexuses were identified and quantified by PGP9.5 staining. To identify the muscular layer of the intestine wall, α-smooth muscle actin staining was used to discriminate myenteric from submucosal plexuses. Unexpectedly, the area occupied by ganglia of the myenteric plexus was significantly increased in SMA mice with more closely-packed neurons compared to control mice (Fig 4). The number of neurons and ganglion density in the myenteric plexuses were significantly greater in SMA compared to control mice, in both duodenum (ganglion density: 256.7±51.6 vs 132.7±10.7, N = 4, P<0.05; neurons: 156.9±16.3 vs 93.8±8.3, N = 6–8, P<0.01) and ileum (ganglion density: 137.9±22.6 vs 63.8±16.0, N = 4, P<0.05; neurons: 89.4±10.9 vs 65.1±3.6, N = 6–8, P<0.05). The ganglion density and number of neurons were significantly reduced to near-normal levels after PMO25 treatment in both duodenum (ganglion density: 256.7±51.6 (SMA) vs 136.5±21.9 (SMA+PMO25), N = 4, P<0.05; neuron number: 156.9± 16.3 vs 65.4± 8.1, N = 8, P<0.001) and ileum (ganglion density: 137.9±22.6 and 71.8±11.0, N = 4, P<0.05; neuron number: 89.4±10.9 vs 59.0±5.4, N = 8, P<0.05). There was no significant difference between all parameters in control and SMA+PMO25 mice (Fig 4).

Bottom Line: We also assessed the therapeutic response of GI histopathology to systemic administration of morpholino antisense oligonucleotide (AON) designed to increase SMN protein expression.Significant anatomical and histopathological abnormalities, with striking reduction of vascular density, overabundance of enteric neurons and increased macrophage infiltration, were detected in the small intestine in SMA mice.We conclude that the observed GI histopathological phenotypes and functional defects observed in these SMA mice are strongly linked to SMN deficiency which can be rescued by systemic administration of AON.

View Article: PubMed Central - PubMed

Affiliation: Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, United Kingdom.

ABSTRACT
Gastrointestinal (GI) defects, including gastroesophageal reflux, constipation and delayed gastric emptying, are common in patients with spinal muscular atrophy (SMA). Similar GI dysmotility has been identified in mouse models with survival of motor neuron (SMN) protein deficiency. We previously described vascular defects in skeletal muscle and spinal cord of SMA mice and we hypothesized that similar defects could be involved in the GI pathology observed in these mice. We therefore investigated the gross anatomical structure, enteric vasculature and neurons in the small intestine in a severe mouse model of SMA. We also assessed the therapeutic response of GI histopathology to systemic administration of morpholino antisense oligonucleotide (AON) designed to increase SMN protein expression. Significant anatomical and histopathological abnormalities, with striking reduction of vascular density, overabundance of enteric neurons and increased macrophage infiltration, were detected in the small intestine in SMA mice. After systemic AON treatment in neonatal mice, all the abnormalities observed were significantly restored to near-normal levels. We conclude that the observed GI histopathological phenotypes and functional defects observed in these SMA mice are strongly linked to SMN deficiency which can be rescued by systemic administration of AON. This study on the histopathological changes in the gastrointestinal system in severe SMA mice provides further indication of the complex role that SMN plays in multiple tissues and suggests that at least in SMA mice restoration of SMN production in peripheral tissues is essential for optimal outcome.

No MeSH data available.


Related in: MedlinePlus