Müller's Muscle as a Sensory Proprioceptive Organ: Histological and Histochemical Analysis

Abstract

Purpose: The purpose of this study was to determine whether proprioceptive nerves are present in Müller's muscle.

Methods: This was a prospective cohort study in which histologic and immunofluorescence analyses of excised Müller's muscle specimens were performed. Twenty fresh Müller's muscle's specimens from patients undergoing posterior approach ptosis surgery in one center between 2017 and 2018 were evaluated by histologic and immunofluorescent analysis. Axonal types were determined by measuring axon diameter in methylene blue stained plastic sections and by immunofluorescence of frozen sections.

Results: We identified large (greater than 10 microns) and small myelinated fibers in the Müller's muscle, with 6.4% of these fibers being large. Immunofluorescent labeling with choline acetyltransferase showed no evidence of skeletal motor axons in the samples, indicating large axons are likely to be sensory and proprioceptive. In addition, we identified C-fibers using double labeling with peripherin and neural cell adhesion molecules.

Conclusions: Overall, large myelinated sensory fibers are present in the Müller's muscle, likely serving proprioceptive innervation. This suggests that proprioception signals from Müller's muscle may have a role in eyelid spatial positioning and retracting, in addition to visual deprivation. This finding sheds new light on our understanding of this complex mechanism.

Figure 1.

Deltoid muscle intramuscular motor nerves and sural nerve fascicle immunofluorescent labeling with anti-peripherin, anti-choline-acetyltransferase (ChAT), and anti-neural cell adhesion molecule (NCAM) immunoglobulins (Ig). These were used as positive controls to validate the immunoglobulins’ specificity. Anti-peripherin Ig stains all axons; anti-ChAT stains structures positive for ChAT, such as cholinergic neurons of motor nerves; and anti-NCAM antibodies stain non-myelinating Schwann cells surrounding unmyelinated axons. (A–C) Control labeling of deltoid muscle showing nerve fibers (anti-peripherin) green labeled axons that are also positive for choline-acetyltransferase (ChAT), indicating that they are motor nerve fibers. A, Anti-peripherin (green) immunoreactive nerve fibers. B, Anti-ChAT immunoreactivity (red). C, Merged view indicates double labeling of all the nerve fibers (yellow-orange). (D, E) Immunofluorescence of a cross-section of a sural nerve fascicule with anti-peripherin (green) in D to identify axons and with co-labeling of neural cell adhesion molecule (NCAM) which labels non-myelinating Schwann cells (red) in E. Unmyelinated sensory fibers are identified as yellow as they are labeled with both peripherin and NCAM, whereas myelinated fibers remain labeled green.

Figure 2.

Hematoxylin and eosin (H&E) staining of the specimens taken from Müller's muscle conjunctival resection procedure, showing smooth muscle cells (yellow arrow) surrounded by connective tissue (white arrow) and lined by epithelial conjunctival cells (pink arrow).

Figure 3.

Immunofluorescent microscopy labeling of a Müller's muscle (MM) specimen, which demonstrates both myelinated and unmyelinated fibers in the MM. (A–C) Anti-peripherin (A, green) Ig stains all axons; anti-neural cell adhesion molecule (NCAM) Ig (B, red) stains non-myelinating Schwann cells. Double labeling demonstrates the presence of unmyelinated axons (C, yellow in merged image). (D, E) Anti-peripherin (D, green) labels all axons; anti-myelin basic protein (E, red) labels myelinating Schwann cells. Double labeling denotes the presence of myelinated axons (F, arrow, yellow in merged image).

Figure 4.

Immunofluorescent microscopy demonstrates labeling of Müller muscle (MM) with anti-peripherin (green) and anti-choline-acetyltransferase (ChAT) (red) immunoglobulins. Anti-peripherin Ig labels all axons; anti-ChAT labels structures positive for ChAT, such as skeletal motor nerves. Green structures represent positive anti-perpherin immunolabeling of axons (green). Note weak ChAT staining in the muscle fibers. None of the axons in this sample, as well as all other samples, were positive for ChAT, ruling out the presence of motor axons.

Figure 5.

Scanned slides depicting cut sections of fixed Müller muscles stained with methylene blue. (A) Cross-section of a nerve bundle between muscle fibers. (B, C) Cross-sections of myelinated nerves with larger diameters (B, 16.92 µm, C, 15.38 µm). As highlighted by the stain, individual axons are encased in myelin. The arrows indicate axons of various sizes (yellow = smaller diameter and orange = larger diameter). In cases of seemingly elongated fibers, which may represent oblique cuts of circular axon profiles, the diameter measurement was performed over the shortest axis rather than the long one; using this rule, the true diameter was obtained. Overall, 580 usable axons were located and measured, with an average diameter of 5.37 micrometers (standard deviation = 2.89).

Figure 6.

Diameter distribution of 580 myelinated axons observed in Müller's muscle samples. The average diameter was 5.37 micrometers (standard deviation = 2.89); 37 axons (6.4%) measured above 10 micrometers (arrow), with the largest being 21.68 micrometer in diameter. The larger axons correspond to A-beta fibers, indicating that they are proprioceptive.