Extraocular muscle characteristics related to myasthenia gravis susceptibility

Abstract

Background: The pathogenesis of extraocular muscle (EOM) weakness in myasthenia gravis might involve a mechanism specific to the EOM. The aim of this study was to investigate characteristics of the EOM related to its susceptibility to myasthenia gravis.

Methods: Female F344 rats and female Sprague-Dawley rats were assigned to experimental and control groups. The experimental group received injection with Ringer solution containing monoclonal antibody against the acetylcholine receptor (AChR), mAb35 (0.25 mg/kg), to induce experimental autoimmune myasthenia gravis, and the control group received injection with Ringer solution alone. Three muscles were analyzed: EOM, diaphragm, and tibialis anterior. Tissues were examined by light microscopy, fluorescence histochemistry, and transmission electron microscopy. Western blot analysis was used to assess marker expression and ELISA analysis was used to quantify creatine kinase levels. Microarray assay was conducted to detect differentially expressed genes.

Results: In the experimental group, the EOM showed a simpler neuromuscular junction (NMJ) structure compared to the other muscles; the NMJ had fewer synaptic folds, showed a lesser amount of AChR, and the endplate was wider compared to the other muscles. Results of microarray assay showed differential expression of 54 genes in the EOM between the experimental and control groups.

Conclusion: Various EOM characteristics appear to be related to the increased susceptibility of the EOM and the mechanism of EOM weakness in myasthenia gravis.

Figure 1. A. Ocular symptoms in PTMG rats.

B. Wet weight of the extraocular muscles (EOM) in the control group and experiment group. C. Level of creatine kinase (CK) in the serum determined by ELISA. *Significant (P<0.05) difference between the control and experimental groups determined by independent two-sample t test.

Figure 2. Acetylcholinesterase-positive endplates of three muscles in the two groups assessed by acetylcholinesterase (AChE) staining.

Scale bar = 50 µm.

Figure 3. Comparision of NMJs from different muscles.

A. Acetylcholinesterase (AChE) staining of three muscles in the two groups. B. Determination of gray value in NMJ by an automatic analyzer (DM2500,Leica,Germany). C. Determination of pixel areas in NMJ by an automatic analyzer. *Significant (P<0.05) difference between control and experimental groups determined by independent two-sample t test. ^aStatistically significant difference between indicated group and EOM group. ^bStatistically significant difference between indicated group and diaphragm group. Scale bar = 20 µm.

Figure 4. Comparision of AChRs in the NMJ of different muscles by fluorescence histochemistry.

A. Acetylcholine receptors (AChRs) labeled with α-bungarotoxin in the neuromuscular junction (NMJ) of three muscles in the two groups. B. Determination of pixel areas in NMJ by an automatic analyzer. C. Determination of gray value in NMJ by an automatic analyzer. *Significant (P<0.05) difference between the control and experimental groups determined by independent two-sample t test. ^aStatistically significant difference between indicated group and EOM group. ^bStatistically significant difference between indicated group and diaphragm group. Scale bar = 10 µm.

Figure 5. Comparision of the NMJ in different muscles by double-staining.

A. SDH and AChR staining in EOM and diaphragm muscle fibers. B. Determination of pixel areas in neuromuscular junction by an automatic analyzer. *Significant (P<0.05) difference between control and experimental groups by independent two-sample t test. ^aStatistically significant difference between indicated group and type I muscle fibers. ^bStatistically significant difference between indicated group and type IIA muscle fibers. Scale bar = 20 µm.

Figure 6. Ultrastructure of acetylcholine receptors labeled with α-bungarotoxin in the neuromuscular junction of three muscles in the two groups.

Scale bar = 2 µm.

Figure 7. The diversity of the neuromuscular junction in three muscles in the two groups.

A. Ultrastructure of the neuromuscular junction (N = nerve endings). B. Western blot analysis for Agrin and SV2 levels in EOM. *Significant (P<0.05) difference between the control and experimental groups determined by independent two-sample t test. Scale bar = 5 µm.