Figure 1.
Effects of anti-MuSK IgG and albuterol on muscle weakness and body weight.
Mice received 15 daily intraperitoneal injections of anti-MuSK-positive patient IgG. Albuterol was delivered continuously via a subcutaneous minipump. (A) Example of typical whole-body weakness in a mouse on day 15 of the anti-MuSK IgG injection series (chin down, flaccid tail, and limb weakness). This mouse was treated with vehicle only. (B) A physically active mouse on day 15 of the anti-MuSK IgG injection series with albuterol treatment (8 mg/kg/day). (C) Pilot dosage trial. Body-weight and weakness grading traces are shown for single mice that received daily injections of anti-MuSK IgG and were treated with 2, 4 or 8 mg/kg/day albuterol, or vehicle. Body weights are shown on the left ordinate while weakness grades are indicated on the lower right ordinate. A minipump delivering either albuterol or vehicle was implanted subcutaneously on day 0 (arrow). The arrowhead indicates a single cyclophosphamide injection to suppress an active immune response to the human IgG. (D) Normalized body weights and weakness grades for mice receiving daily injections of anti-MuSK IgG and treatment with 8 mg/kg/day albuterol (filled squares) or vehicle (open circles). Error bars in panel D represent the mean ± SEM for n = 4 mice in each treatment group (*P<0.05, unpaired Student’s t test).
Figure 2.
Effects of anti-MuSK IgG and albuterol treatment upon AChR staining intensity.
Transverse sections of the diaphragm and tibialis anterior muscles collected on day 15 of the IgG injection series were stained for motor endplate AChR with Alexa488-α-bungarotoxin. (A) Typical bright, crescent-shaped AChR staining of two endplates in the diaphragm muscle of a healthy naive mouse (no treatment). Scale bar = 20 µm. (B–C) Dim AChR staining of endplates from mice that received injections of anti-MuSK IgG and were treated with either vehicle (B) or 8 mg/kg/day albuterol (C). (D) Intensity of endplate staining for AChR (mean pixel intensity) in the tibialis anterior muscle of naive mice and mice that received 15 daily injections of anti-MuSK IgG, with or without albuterol treatment (8 mg/kg/day). (E) Intensity of endplate AChR staining in the diaphragm muscle of the same mice. (F) Counts of the number of AChR-stained endplates per microscope field in the diaphragm muscle of healthy naive mice, or mice injected with anti-MuSK IgG and treated with albuterol (8 mg/kg/day), or vehicle. Counts were made by an operator who was blind to the treatment group of the photomicrographs. Data in D, E and F represent the mean ± SEM for n = 3 mice (*P<0.05, **P<0.01, ***P<0.001; one-way ANOVA with Bonferroni’s multiple comparison post-test).
Figure 3.
Longitudinal enface images of motor endplates in the diaphragm muscle of mice after injections of anti-MuSK IgG and albuterol treatment.
(A) Three exemplar NMJs in a healthy naive mouse, double labeled for AChR (red) and nerve (synaptophysin and neurofilament; green), illustrating typical large, pretzel-shaped AChR plaques each covered by nerve terminal staining. (B) NMJs from a mouse that received injections of anti-MuSK IgG and treatment with vehicle. Endplate AChR staining was weak and AChRs were fragmented into multiple small AChR clusters. (C) NMJs from a mouse that received injections of anti-MuSK IgG and treatment with albuterol (8 mg/kg/day). AChR clusters were less fragmented compared to those of vehicle-treated mice. Some of the nerve terminals appeared better aligned with AChRs. Scale bar is 20 µm. (D–G) Quantitative comparison of endplates from mice treated with either 8 mg/kg/day albuterol (black bars) or vehicle (grey bars) at day 15 of the anti-MuSK IgG injection series. (D) Average number of AChR clusters per endplate (E) Percentage of endplate AChRs covered by nerve and percentage of endplate nerve terminal staining with AChRs clustered beneath it. (F) Area of synaptic specialization at the endplate: AChR, nerve and AChR-nerve overlap. (G) Relative intensity of endplate AChR staining and nerve staining. Bars represent the mean SEM for n = 3 mice in each treatment group (*P<0.05).
Figure 4.
Albuterol treatment did not prevent neuromuscular failure in mice injected with anti-MuSK IgG.
(A) A representative compound muscle action potential (CMAP) trace recorded from the gastrocnemius muscle of a mouse after 15 daily injections of anti-MuSK IgG. The amplitude is seen to decline during repetitive stimulation of the sciatic nerve (3 stimuli per second). (B) Average decrement in CMAP amplitude for myasthenic mice treated with either albuterol (8 mg/kg/day; filled squares) or vehicle (open circles) on day 15 the anti-MuSK IgG injection series. The mean amplitude of the first CMAP of the train was 9.6±2.7 mV for mice injected with anti-MuSK IgG and treated with albuterol compared to 13.5±2.1 mV for mice injected with anti-MuSK IgG and treated with vehicle (P>0.05). Data represent the mean ± SEM for n = 3–4 mice in each treatment group.
Figure 5.
Albuterol did not improve endplate potentials in mice injected with anti-MuSK IgG.
Comparison of endplate potentials (EPPs) for myasthenic mice treated with either 8 mg/kg/day albuterol or vehicle. Recordings were made from the phrenic nerve-diaphragm muscle preparation on day 15 of the anti-MuSK IgG injection series. (A) Amplitude of nerve-evoked EPPs from mice injected with anti-MuSK IgG and treated with albuterol (8 mg/kg/day; black bar) or vehicle (grey bar). (B) Amplitudes of spontaneous mEPPs from the same mice. (C) Frequency of mEPPs. (D) Quantal content. Data in panels A–D represent the mean ± SEM for n = 3 mice in each treatment group (unpaired Student’s t-test). None of these parameters differed significantly between albuterol- and vehicle-treatment groups. (E) Scatter plot of quantal content versus mEPP amplitude for endplates in the diaphragm muscle of anti-MuSK-injected mice that were treated with vehicle (each endplate is represented by a filled circle). Data were fitted by linear regression. No significant correlation was found (P = 0.69). Probability values (P) reflect the likelihood that the slope was non-zero. (F) Quantal content versus mEPP amplitude for endplates of anti-MuSK-injected mice that were treated with albuterol (P = 0.43).
Figure 6.
Cross-sectional area of muscle fibers.
(A–C) Frequency distributions of muscle fiber cross-sectional areas for the diaphragm muscle. (D–F) Cross-sectional areas for fibers from the tibialis anterior muscle. Data were pooled from healthy naive mice (A & D; n = 5 mice) or mice that received 15 daily injections of anti-MuSK IgG together with vehicle (B & E; n = 4 mice) or 8 mg/kg/day albuterol (C & F; n = 4 mice).