SMΝΔ7 mice show breathing and airflow defects with significant pathology of respiratory and oral tract tissues

Figures

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  Figure 1. SMNΔ7 mice showed respiratory deficits as quantified by plethysmography. Smn+/+ mice are represented as gold bars, and SMNΔ7 mice are represented as peach bars. (A) Waveforms of P7 mice. (B–E) P7 plethysmography parameters are located on top (head-out plethysmography), and P14 parameters are located on the bottom (whole-body plethysmography). Respiratory conditions for these studies were N, normoxia; HC, hypercapnia; HC + HX, hypercapnia + hypoxia. (B) Frequency measured as breaths per minute. P14 Smn+/+ vs. SMNΔ7 HC + HX *** p = 0.0009. P14 Smn+/+ N to HC ** p = 0.0097. (C) Tidal volume (mL/gram) normalized to weight. P7 Smn+/+ vs. SMNΔ7 HC + HX * p = 0.0102. P7 Smn+/+ N to HC * p = 0.0240, N to HC + HX **** p = <0.0001, HC to HC + HX * p = 0.0245. P14 Smn+/+ vs. SMNΔ7 N *** p = 0.0003, HC ** p = 0.0025. P14 Smn+/+ N to HC * p = 0.0104, HC to HC + HX ** p = 0.0097. (D) Peak inspiratory flow is measured as mL/s. P7 Smn+/+ vs. SMNΔ7 N ** p = 0.0014, HC *** p = 0.0002, HC + HX **** p = <0.0001. P7 Smn+/+ N to HC + HX *** p = 0.0009, HC to HC + HX * p = 0.0256. P14 Smn+/+ vs. SMNΔ7 HC and HC + HX **** p = <0.0001. P14 Smn+/+ N to HC and N to HC + HX **** p = <0.0001. (E) Peak…

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  Figure 2. SMNΔ7 mice diaphragm muscle fiber type populations were comparable to wild-type mice. Smn+/+ mice are represented as gold bars, and SMNΔ7 mice are represented as peach bars. (A) Representative 10X images of cross sections of P7 diaphragms of Smn+/+ and SMNΔ7 mice. Laminin is represented as white, type I fibers are blue, type 2A fibers are red, and type 2B fibers are green. (B) Representative images of the cross-section of P14 diaphragms. (C) Diaphragm muscle fiber populations of P7 Smn+/+ and SMNΔ7 mice. No significant changes found in any population. (D) Diaphragm muscle fiber populations of P14 Smn+/+ and SMNΔ7 mice. No significant changes found in any population. P7 Smn+/+ n = 4, SMNΔ7 n = 5. P14 n = 5 for each group. Quantification of fiber areas found in Table 1.

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  Figure 3. SMNΔ7 oral cavity and internal intercostal muscles showed significant changes in NMJ innervation. Smn+/+ mice are represented as gold bars, and SMNΔ7 mice are represented as peach bars. (A) Representative images of P7 mylohyoid NMJs taken at 40X. Green represents neurofilament heavy (NF-H) and synaptic vesicle 2 (SV2), while red represents acetylcholine receptors (AChR). (C,D) NMJ quantification as a percentage of analyzed endplates as FI = fully innervated, PI = partially innervated, and FD = fully denervated. (B) Cartoon representing oral cavity muscles. (C) Quantification of lower respiratory cavity NMJ innervation status. Internal intercostal FI * p = 0.0170. (D) Quantification of oral cavity and upper respiratory tract NMJ innervation status quantification. Masseter FI *** p = 0.0002, FD * p = 0.0479. Mylohyoid FI * p = 0.0206. Genioglossus+geniohyoid FI ** p = 0.0016, PI ** p = 0.0032. N = ≥4 for all groups.

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  Figure 4. Phrenic and hypoglossal nerves show significant morphological changes. (A) Representative images of P7 phrenic nerves. 1 μm thick cross sections stained in toluidine blue and imaged at 100X. (B) Representative images of P7 hypoglossal nerves. 1 μm thick cross sections stained in toluidine blue and imaged at 100X. Quantification of nerves is found in Table 2 (phrenic) and 3 (hypoglossal). Phrenic nerve P7 Smn+/+ n = 4 SMNΔ7 n = 6, P14 n = 5 per group. Hypoglossal nerve P7 Smn+/+ n = 4 SMNΔ7 n = 5, P14 Smn+/+ n = 4 SMNΔ7 n = 5.

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  Figure 5. SMNΔ7 lung H&E shows morphological changes at P14. (A,B) Representative images of lungs that were paraffin-embedded and H&E-stained. (A) P7 lungs. (B) P14 lungs. The black arrow points to the collapse of airspaces (atelectasis), and the yellow arrow points to hemorrhage. Quantification of lung tissues is found in Table 4; P7 n = 4 per group, P14 n = 5 per group.