3D Micro- and Nanostructural Analysis of Connective Tissue in the Human Optic Nerve Head (ONH)

Abstract

 

Purpose:

 

To develop techniques to analyse the 3D micro- and nanoarchitecture of connective tissue within the human lamina cribrosa (LC) and adjacent tissues.

 

Methods:

 

Two-photon fluorescence (TPF) (_ex=800nm, _em≈500nm) and second harmonic generation (SHG) (_ex=800nm, _em=400nm) imaging were performed on 100µm ONH cryosections from pre- to postlaminar regions (n=10 sections per ONH). Non-linear signals were validated against Masson’s Trichrome Green and immunolabelling. Collagen to pore ratio was calculated and collagen and elastic fiber orientation were evaluated in 3D datasets. Small angle X-ray scattering (SAXS; beam size 12x19μm, =0.09nm, specimen-to-detector distance 0.9m) was used to analyse elastic fiber, collagen and myelin nanoarchitecture in each ONH section on station I22 (Diamond Light Source, UK).

 

Results:

 

Masson’s Trichrome Green and immunolabelling of collagen types I, III and IV and elastin validated the derivation of SHG and TPF signals from fibrillar collagen and elastic fibers, respectively. These stretched radially across the optic canal in the LC, encircled the central retinal vessels and were absent in the prelamina. In post laminar ON septae, collagen was perpendicular to that in the LC. 3D reconstructions of the ONHs were produced from the non-linear signals (see figure). Collagen to pore ratios showed collagen-related SHG scattering to be greatest in the LC (1.90) with lower values in the prelamina (0.45) and postlaminar ON (0.66). SAXS patterns revealed the 67nm fibrillar axial structure of collagen and a 3.84nm reflection for elastin. Additionally X-ray peaks indexed on 16nm representative of CNS myelin in the postlaminar ON.

 

Conclusions:

 

Non-linear microscopy and SAXS enable elucidation of collagen and elastin micro- and nanoarchitecture, respectively. 3D analysis of ONH architecture is essential to our understanding of ONH biomechanics in order to determine those individuals predisposed to glaucomatous optic neuropathy.  

 

Keywords: lamina cribrosa • extracellular matrix • imaging/image analysis: non-clinical