Order In The Chaos: Regions Of Highly Aligned Radial Collagen Fibers In The Peripapillary Sclera (PPS)

Abstract

 

Purpose

 

To map the architecture of the PPS collagen fibers with fiber-scale resolution over a wide area and identify major organizational components.

 

Methods

 

Eight eyes from 4 sheep and 3 eyes from 3 humans were analyzed. Eyes were fixed at IOP=5mmHg in 10% formalin. Circular regions of posterior pole centered on the ONH 8 to 14mm diameter were excised and cryosectioned coronally (30µm thickness). An average of 75 sections per eye (range 42-154) from extraocular optic nerve to the vitreous were imaged with a microscope (SMZ1500, 16bit grayscale, 4.1µm/pixel) and local collagen fiber orientation was determined [Sigal ARVO 2013]. Areas representative of three regions were manually identified in every image (where visible): the PPS immediately adjacent to the scleral canal, the innermost sclera and a randomly selected region of PPS elsewhere (Figure 1). For each marked area (674 total) we computed the mean and SD of fiber orientation (radial to circumferential) relative to the center of the scleral canal. A linear mixed effects model was fit to test if mean angle and angle variations were significantly predicted by region accounting for correlations within eye and individual. In selected regions fiber orientation and splay were confirmed using confocal microscopy and polarized light microscopy at higher magnification (0.8µm/pixel).

 

Results

 

Three distinct regions of collagen fiber orientation were consistently discernible in all eyes of both species (Figure). Area mean orientations were significantly different between all regions (p<0.0001), with strong alignments in the radial and ring regions and isotropic orientations in the random region. The layer of radially oriented fibers was estimated to be 60-120µm thick in sheep and 30-90µm thick in human, extending at least 3mm from the canal.

 

Conclusions

 

The most important finding is that we identified a new subpopulation of collagen fibers in the innermost sclera highly aligned radially from the canal. We have also presented the first measurements of the high degree of fiber alignment in the ring around the canal, which had been suspected, but not measured. These structures may stabilize the posterior pole to protect the tissue from mechanical insult.

 

 

Fig. 1. Fiber-trace image of a human eye showing areas selected for each region type

 

 

Fig 2. (A) Means and (B) standard deviations in fiber orientation by region and species.

 

Keywords: 708 sclera • 577 lamina cribrosa • 599 microscopy: light/fluorescence/immunohistochemistry