Meibomian Gland Probing Stimulates a Proliferative Epithelial Response Resulting in Duct Regeneration

Abstract

Purpose: To demonstrate that the meibomian gland ductal basement membrane and basal epithelial cell layer are in continuity with and may derive from lid margin orifice-associated rete ridge epithelial/basement membrane structures (OARREBS) and to characterize changes in the distal duct microanatomy after meibomian gland probing (MGP) using in vivo confocal microscopy (IVCM).

Patients and methods: Pre/post-MGP IVCM examinations were performed on upper lids. Thirty-six identical glands from 20 lids of 16 patients (49.24 ±17.11 y/o with 13:3 F:M) were identified, analyzed, and compared to control cases. Statistical analyses were performed using ImageJ software and IBM SPSS version 27. All MGPs were performed within 12 weeks of the initial examination. Post-MGP follow-up exams occurred at 5.03 ±4.48 months.

Results: Post-MGP images showed more superficially organized OARREBS with accelerated and more superficial basement membrane formation, and an average increase of 32.2%, 25.4%, 32.04%, 77.7%, and 81.3% in duct wall epithelial cell layers (DWECL) (p < 0.001, compared to control (CTC) p < 0.001), distal duct wall thickness (DWT) (p < 0.001, CTC p < 0.001), proximal DWT (p < 0.001, CTC p < 0.001), distal lumen area (p < 0.001, CTC p = 0.037), and proximal lumen area (p < 0.001, CTC p = 0.007), respectively. The increase in the distal DWT and lumen area correlated with the months of follow-up (p = 0.004 and p = 0.010, respectively). Immediate post-MGP imaging revealed the probe track confined to the ductal epithelial compartment.

Conclusion: MGP appears to stimulate a proliferative epithelial response characterized by an accelerated more superficial formation of ductal basement membrane with increased DWECL as well as DWT and lumen area at two separate duct foci. These findings suggest activation of lid margin meibomian gland precursor cells and confirm that MGP stimulates an epithelial regenerative phenomenon, not a fibrotic one.

Keywords: dry eye; meibomian gland dysfunction; meibomian gland probing; meibomian gland regeneration; precursor cells; rete ridges.

Figure 1

Image of rete ridges at the lid margin of an upper lid using in vivo confocal microscopy (IVCM). (A) Superficial epithelial cell aggregates which organize into orifice-associated rete ridge epithelial/basement membrane structures (OARREBS) with increased depth. (B) At a greater depth, each rete ridge has a bright outer epithelial cell layer (blue bracket), a dark center dermal component (green asterisk), and a junctional basement membrane that resides between these two (pink arrow)., White dashed circle denotes the area of interest while red circle highlights a single rete ridge from the progression of epithelial cell aggregate (A) to complete rete ridge formation (B) as the depth increases. Scale bar, 50µm.

Figure 2

Images of progressive volumetric scans of lid margin rete ridges in continuity with Meibomian gland basement membrane and basal epithelium using in vivo confocal microscopy (IVCM). Each meibomian gland orifice (A–D) demonstrates individual orifice associated rete ridges in peri-lumenal position (white asterisks) ovalize, flatten, and appear to coalesce with adjacent rete ridges to form the Meibomian gland basement membrane and ductal basal epithelium with increased depth (white bracket). Scale bar, 50µm.

Figure 3

(A and B) Images of progressive volumetric scans of an identical gland pre and post-Meibomian gland probing (MGP) using in vivo confocal microscopy (IVCM). Red outline indicates inlay of larger image. (A) Meibomian gland pre-MGP showing orifice-associated rete ridge epithelial/basement membrane structures (OARREBS) (white circle) in continuity with the basement membrane and basal epithelium of the Meibomian gland. (B) Identical Meibomian gland to (A) post-MGP showing the same OARREBS (white circle) with a single epithelial cell (white arrow) cleaving from the OARREBS and incorporating into the Meibomian gland duct wall epithelial cell layers. The remaining smaller OARREBS is further from the Meibomian gland lumen with subsequent morphologic change into a portion of the basement membrane. Large image scale bar, 50µm; small image scale bar, 20µm. (C–E) IVCM images immediately before and after MGP using a 76μm probe. (C) Meibomian gland immediately pre-MGP. (D and E) Meibomian gland immediately post MGP at a depth of 31μm and 53μm, both showing a notch at the lumen inferiorly. Image (D) demonstrates apparent debridement of terminally differentiated and keratinized epithelium lining the lumen while (E) shows probe path through suprabasal ductal epithelium. Probing procedure is confined to the epithelium compartment, explaining lack of secondary fibrosis. (F) Location and presence of a 4mm length probe (76μm OD) inside the duct of a meibomian gland using infrared video meibography. Note that acinar-ductule units are visible overlying the device. (F) adapted with permissions from Maskin, Steven L., and Sreevardhan Alluri. “Meibography guided intraductal meibomian gland probing using real-time infrared video feed”. British Journal of Ophthalmology 104.12 (2020): 1676–1682.

Figure 4

Graphs demonstrating the change in each parameter from pre-Meibomian gland probing (MGP) (red) to post-MGP (blue). (A) Meibomian glands show an average of 17.13% more superficial appearance of rete ridges post-MGP (t(30)=4.597, p<0.001). (B) Meibomian glands had an average of 14.22% earlier basement membrane formation post-MGP (t(36)=5.03, p<0.001). (C) Meibomian glands demonstrate an average acceleration compared to baseline of 9.79% from rete ridges (RR) to basement membrane (BM) post-MGP (t(27)=3.97, p=0.002). (D) Meibomian glands had an average increase in duct wall epithelial cell layers (DWECL) of 32.20% post-MGP (t(31)=4.68, p=<0.001). (**p<0.01, ***p<0.001).

Figure 5

(A) Representative in vivo confocal microscopy (IVCM) images of an identical Meibomian gland at a depth of 81µm pre and post-Meibomian gland probing (MGP). This example is 0.46 months post-MGP. Statistical analysis revealed a significant 25.40% average increase in distal duct wall thickness (DWT) (purple) post-MGP (t(36)=5.035, p<0.001) and a significant 77.74% average increase in distal lumen area (blue) post-MGP (t(36)=5.035, p<0.001). (B) Representative IVCM images of the same identical Meibomian gland as “A” at a depth of 110µm pre and post-MGP. Statistical analysis revealed a significant 32.04% average increase in proximal DWT (purple) post-MGP (t(30)=4.597, p<0.001) and a significant 81.34% average increase in proximal lumen area (blue) post-MGP (t(30)=4.597, p<0.001). (C) Average percent increase in distal (blue) and proximal (red) DWT post-MGP over time (months). When compared to baseline, glands with a less than 1 month, 1 month or greater and less than 3 months, 3 months or greater and less than 6 months, 6 months or greater and less than 9 months, 9 months or greater and less than or equal to 13 months follow-up had an average increase in distal DWT of 33.59% (n=9, p=0.015), 34.60% (n=9, p=0.008), 19.64% (n=4, p=0.068), 19.86% (n=7, p= 0.028) and 11.89% (n=7, p=0.063), respectively, and an average increase in proximal DWT of 34.71% (n=9, p=0.011), 38.75% (n=8, p=0.012), 35.70% (n=2, p=0.180), 27.12% (n=6, p=0.028), and 20.91% (n=5, p=0.080), respectively. (D) Average percent increase in distal (blue) and proximal (red) lumen area post-MGP over time (months). When compared to baseline, glands with a less than 1 month, 1 month or greater and less than 3 months, 3 months or greater and less than 6 months, 6 months or greater and less than 9 months, 9 months or greater and less than or equal to 13 months follow-up had an increase in distal lumen area of 120.95% (n=9, p=0.011), 158.56% (n=9, p=0.008), 20.59% (n=4, p=0.273), 17.10% (n=7, p=0.128), and 11.56% (n=7, p=0.237), respectively, and an increase in proximal lumen area of 67.52% (n=9, p=0.021), 169.19% (n=8, p=0.017), 39.49% (n=2, p=0.180), 37.83% (n=6, p=0.028), and 34.68% (n=5, p=0.043), respectively. Scale bar, 50µm. (*p<0.05, **p<0.01, ***p<0.001).