Histologic validation of optical coherence tomography-based three-dimensional morphometric measurements of the human optic nerve head: Methodology and preliminary results

Abstract

Purpose: To compare the three-dimensional (3D) morphology of the deep load-bearing structures of the human optic nerve head (ONH) as revealed in vivo by spectral domain optical coherence tomography (SDOCT) with ex vivo quantitative 3D histology.

Methods: SDOCT imaging of the ONH was performed in six eyes from three brain-dead organ donors on life-support equipment awaiting organ procurement (in vivo conditions). Following organ procurement (ex vivo conditions), the eyes were enucleated and underwent a pars plana vitrectomy followed by pressurization to physiologic IOP and immersion fixation. Ex vivo ONH morphology was obtained from high-fidelity episcopic fluorescent 3D reconstruction. Morphologic parameters of the observed ONH canal geometry and peripapillary choroid, as well as the shape, visibility and depth of the lamina cribrosa were compared between ex vivo and in vivo measurements using custom software to align, scale, and manually delineate the different regions of the ONH.

Results: There was significant correspondence between in vivo and ex vivo measurements of the depth and shape of the lamina cribrosa, along with the size and shape of Bruch's membrane opening (BMO) and anterior scleral canal opening (ASCO). Weaker correspondence was observed for choroidal thickness; as expected, a thinner choroid was seen ex vivo due to loss of blood volume upon enucleation (-79.9%, p < 0.001). In addition, the lamina was shallower (-32.3%, p = 0.0019) and BMO was smaller ex vivo (-3.38%, p = 0.026), suggesting post mortem shrinkage of the fixed tissue. On average, while highly variable, only 31% of the anterior laminar surface was visible in vivo with SDOCT (p < 0.001).

Conclusions: Morphologic parameters by SDOCT imaging of the deep ONH showed promising correspondence to histology metrics. Small but significant shrinkage artifact, along with large effects of exsanguination of the choroid, was seen in the ex vivo reconstructions of fixed tissues that may impact the quantification of ex vivo histoarchitecture, and this should be considered when developing models and biomarkers based on ex vivo imaging of fixed tissue. Lack of visibly of most of the lamina surface in SDOCT images is an important limitation to metrics and biomarkers based on in vivo images of the ONH deep tissues.

Keywords: Glaucoma; Histology; Optic nerve head morphology and biomechanics; Spectral domain optical coherence tomography.

Figure 1.

Custom software to delineate ONH anatomy of the same brain-dead organ donor eye in both in vivo (before organ recovery by optical coherence tomography; OCT) and ex vivo conditions (after 3D reconstruction with episcopic autofluorescence imaging). In the OCT B-scan (upper plot), The Bruch’s membrane and opening (BMO) are marked in orange; anterior scleral surface and opening (ASCO) is marked in yellow; anterior lamina cribrosa surface (ALCS) is marked in fuchsia. In the digital section of the 3D histology reconstruction representing the same location in the ONH depicted in the OCT B-scan above. In the histology, additional landmarks of the retina are visible and delineated: the posterior lamina cribrosa surface is marked in red; pia mater is marked in green; posterior scleral surface in blue; internal surface of the dura mater in cyan. Internal limiting membrane marked in gray.

Fig. 2.

Representation of the three-dimensional fitting of the Bruch’s membrane (BM) (in green; plot A) and anterior scleral points (in yellow; plot B). Plot C: BM and anterior sclera points belonging to a 250-μm-wide anulus radially distant 1500μm from the optic nerve head center was used to compute BM- and sclera-based reference planes and choroidal thickness.

Fig. 3.

In red, representation of the three-dimensional paraboloid fitting of the anterior lamina cribrosa surface (ALCS). In white, 500-μm-radius cylinder perpendicular to the BMO reference plane (in green), and passing for the BMO centroid (dark green). The intersection between the cylinder and ALCS defined the central portion of the ALCS for which ALCS depth is computed. Depth was computed as the average distance between the central ALCS and the BMO reference plane.

Fig. 4.

En face views of the visible anterior lamina cribrosa surface (ALCS, in red) delineation points, anterior scleral canal opening (ASCO, in yellow) area, and Bruch’s membrane opening (BMO, in green) area. Left plot: Visibility of these regions in the ex vivo episcopic autofluorescence histology images. Right plot: Visibility of these regions in the vivo optical coherence tomography images. Notice the much smaller visibility of ALCS in vivo.

Fig. 5.

Bland-Altman plots with limit of agreement (LoA) of BMO major axis (left plot), BMO minor axis (center plot), and BMO area (right plot). (“N”: normal eyes; “G”: glaucoma eyes.)

Fig. 6.

Bland-Altman plots with limit of agreement (LoA) of ASCO major axis (left plot), ASCO minor axis (center plot) and ASCO area (right plot).

Fig. 7.

Left plot: Bland-Altman and limit of agreement (LoA) for choroidal thickness showed a thicker choroid in the in vivo measures. Right plot: anterior lamina cribrosa surface (ALCS) visibility area LoA showed a large reduction of visibility in the in vivo measures compared to ex vivo.

Fig. 8.

Bland-Altman plots with limit of agreement (LoA) of the ALCS distance from: the BMO reference plane (top-left); the BM reference plane (top-right); ASCO reference plane (bottom-left); sclera reference plane (bottom-right).

Fig. 8.

Bland-Altman plots with limit of agreement (LoA) of the ALCS distance from: the BMO reference plane (top-left); the BM reference plane (top-right); ASCO reference plane (bottom-left); sclera reference plane (bottom-right).

Fig. 9.

Bland-Altman plots with limit of agreement (LoA) of BMO maximum curvature (left plot), minimum curvature (center plot) and global shape index (right plot).