Micro-computed Tomography (µCT) for the Structural Analysis of the Lamina Cribrosa (LC)

Abstract

 

Purpose

 

To image the beam microarchitecture of the LC at various intraocular pressure (IOP) levels. Such information can be used to determine LC mechanical properties, of interest in glaucoma.

 

Methods

 

Porcine and human eyes were obtained and prepared within 24 h of death. The posterior poles were glued to a pressurization chamber and inflated overnight using a tissue staining solution at 15 mmHg to enhance X-ray absorption in µCT. Contrast agents included iodine/potassium iodide (IKI) and 2% phosphotungstic acid (PTA). µCT scans were performed in a conventional system (SCANCO Medical µCT 50, 90 keV, 155 mA, 1300 projections, 1s exposure time (ET), 5 µm isotropic resolution (IR)). The collagen autofluorescence signal was collected using multiphoton microscopy (Zeiss LSM 710 NLO) in order to validate that µCT accurately resolved the LC beams. Untreated posterior poles were subjected to high-resolution phase contrast µCT at the UK Diamond Light Source synchrotron (53 keV, 300 mA, 900 projections, 1.2s ET, 1µm IR).

 

Results

 

Overnight PTA staining under pressure provided high contrast between neural and beam tissue (Fig. a), but markedly increased the stiffness of the sclera and LC. Multiphoton microscopy on different specimens resolved similar beam microarchitecture, suggesting that PTA binds to the LC beams (Fig. b). In contrast, IKI enhanced the LC pores and did not provide sufficient contrast for LC beam detection (Fig. c). Phase-contrast µCT resolved beams with high contrast without need for a staining agent (Fig. d).

 

Conclusions

 

Conventional µCT with tissue staining is a robust tool to detect LC beam microarchitecture. However, the stain-induced tissue stiffening prevents this method being used for physiologically relevant deformation measurements. We are screening a non-stiffening contrast agent (collagen-targeting gold nanoparticles) and optimizing the experimental protocol for phase-contrast µCT to obtain measurements of IOP-induced LC deformation.

 

 

(a) Cross-section of a conventional µCT scan after overnight pressure-staining with 2% PTA. (b) Cross-section of a multiphoton microscopy image on a different specimen. (c) Cross-section of a conventional µCT scan after overnight pressure-infusion staining with 1% IKI. (d) Cross-section of a phase-contrast µCT scan, requiring no tissue staining. All images are from porcine LC (average diameter 3.5 mm).

 

Keywords: 577 lamina cribrosa • 552 imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • 551 imaging/image analysis: non-clinical