Immunofluorescent Triple-Staining Technique to Identify Sensory Nerve Endings in Human Thumb Ligaments

Abstract

Ligament innervation purportedly plays a critical role in stability, proprioception and pathology of joints with minimal bony constraints. The human thumb carpometacarpal (CMC) joint is such a joint: with a complex saddle configuration and wide circumduction, its constraint is primarily ligamentous and it is prone to osteoarthritis. CMC reconstruction is the most commonly performed arthritis surgery in the upper extremity. Little, however, is known about CMC ligament innervation. We describe a novel triple-staining immunofluorescence technique using the markers for low-affinity neurotrophin receptor p75, the pan-neuronal marker protein gene product (PGP) 9.5 and 4′,6′-diamidino-2-phenylindole (DAPI) to simultaneously detect and differentiate between specific sensory nerve endings: the Pacini corpuscles, the Ruffini endings and nerve fascicles. Five primary CMC ligaments (dorsal radial, dorsal central, posterior oblique, anterior oblique and ulnar collateral ligaments) were harvested from 10 fresh-frozen human cadaver hands. Following paraffin sectioning, each ligament was stained using a triple-stain technique and imaged with fluorescence microscopy. Multidimensional acquisition permitted simultaneous capture of images at different wavelengths. Pacini corpuscles were distinguished by their distinct p75 immunoreactive capsules, and Ruffini endings by their overlapping p75 and PGP9.5 immunoreactive dendritic nerve endings. Simultaneous use of PGP9.5, p75 and DAPI immunofluorescence to analyze innervation patterns in human ligaments provides descriptive analysis of staining patterns and receptor structure as well as clues as to the proprioceptive function of CMC ligaments and the joint as a whole. Our novel findings of CMC ligament innervation augment the study of normal and pathological joint mechanics in this joint so prone to osteoarthritis.

Ultrastructure and Innervation of Thumb Carpometacarpal Ligaments in Surgical Patients With Osteoarthritis

“…As previously reported in cadavers, Pacini corpuscles were identified through their onion-like, lamellar capsule with p75 staining and lacking PGP9.5 and DAPI immunofluorescence (Fig. 3) [26].…”

“…As previously reported in cadavers, Ruffini endings were distinguished by their coiled dendritic endings that stain independently with p75 and PGP9.5 and also overlap, emitting yellow-orange fluorescence (Fig. 2) [26]. They range in size from 50 to 150 lm.…”

“…This has been shown in normal cadaveric dissections [16,24,26] but the characteristics in surgical patients are unknown. (2) Are sensory nerve endings, ie, mechanoreceptors, present in the AOL and DRL of patients with CMC-1 OA, and, if so, is there a difference in mechanoreceptor types between the AOL and DRL, respectively?…”

“…ProLong Gold Anti-Fade Reagent with 49,69-diamidino-2-phenylindole (DAPI) (Invitrogen) was used concomitantly to highlight nuclear material in specimens. In conjunction, triple staining with p75, PGP9.5, and DAPI is a recently validated technique for visualizing mechanorecptors, nerves, and arteries/arterioles in contrast with collagen within ligaments [26].…”

“…An ordinal grading system previously used for analysis of ligament innervation was used to quantify the degree of innervation and to assess for mechanoreceptor presence [15,26]. The grading system ranged from (+++) to (À) with (+++) representing the presence of several nerve fascicles and mechanoreceptors, (++) representing the presence of a single nerve fascicle and receptor, (+) representing the presence of nerve fascicles but no receptors, and (À) representing the presence of no nerve fibers, fascicles, or mechanoreceptors.…”