Quantification of Optic Nerve Cross Sectional Area on MRI: A Novel Protocol using Fiji Software

Summary

We provided a detailed protocol for a standardized method of optic nerve assessment and quantification using MRI, utilizing a widely available imaging sequence, and open access software for image analysis. Following this standardized protocol would provide meaningful data for comparison between different patients and different studies.

Abstract

Optic nerve assessment is an important aspect of glaucoma diagnosis and follow-up. This project describes a protocol for a unified methodology of optic nerve cross-sectional assessment and quantification using 3 T MRI for image acquisition and ImageJ's Fiji software for image processing quantification. Image acquisition was performed using 3 T MRI, with proper instructions for the patient to ensure straight fixation during imaging. A T2-weighted fat suppressed sequence was used. A coronal cut taken 3 mm behind the globe and perpendicular to the optic nerve axis should be uploaded to the software. Using the threshold function, the white matter area of the optic nerve is selected and quantified, thus, eliminating inter-individual measurement bias. We also described the normal limits for the optic nerve cross-sectional area according to age, based on previously published literature. We used the described protocol to assess optic nerve of a suspected glaucoma patient. The optic nerve cross-sectional area was found to be within the normal limits, a finding further confirmed via optical coherence tomography of the optic nerve.

Introduction

Glaucoma is an optic neuropathy that is considered to be the most common cause of irreversible blindness¹. Despite that, it is still poorly understood in terms of its pathophysiology and diagnosis, with no single standard reference for establishing the diagnosis². According to the National Institute for Health and Care Excellence (NICE) the diagnosis of primary open-angle glaucoma (POAG) requires the assessment of multiple domains, including optic disc assessment on fundus exam or optical coherence tomography (OCT) imaging, visual field assessment, and intraocular pressure measurement ³. The idea behind diagnosing glaucoma is establishing the presence of progressing optic neuropathy, which can be done quantitively on OCT⁴. In this regard, MRI can also be used for optic nerve assessment and quantification of its white matter area⁵, but for this to be clinically meaningful, the protocol used in optic nerve white matter quantification needs to be standardized. Moreover, a protocol should also accommodate inter-individual variation, a factor that might affect accuracy in different diseases⁶.

Optic nerve assessment in glaucoma is optimally assessed via ophthalmic imaging, including OCT, where the most anterior part of optic nerve (e.g., optic disc) is assessed. On the other hand, the use of MRI for optic nerve assessment usually assesses the retrobulbar part of optic nerve at various distances from globe. Several studies found a strong correlation between optic disc assessment using OCT and MRI^7,8. However, there is still no unified protocol for optic nerve assessment and quantification on MRI. Outlining the optic nerve border on MRI has been used to quantify its cross-sectional area⁵. However, this method has considerable inter-rater variability, as it needs to be done by an experienced rater and requires considerable time for outlining. The aim of the current project was to provide a protocol for a unified methodology for optic nerve cross sectional assessment and quantification using 3 T MRI for image acquisition and ImageJ's Fiji software for image processing and quantification.

Protocol

The following study was approved by the research committee and institutional review board of the University of Jordan Hospital. The following protocol will describe the imaging technique used to acquire MRI images, followed by image processing and optic nerve quantification using Fiji software.

1. MRI image acquisition

NOTE: MR image acquisition was done using a 3 Tesla (3 T) MRI to perform multiplanar T2-weighted fat suppression sequence (Table of Materials).

1. Fully explain the examination to the patient. The following include instructions and explanations that need to be mentioned to the patient.
   1. Explain to the patient that they will need to change clothes and wear a special gown for imaging.
   2. Have patients remove any worn eyeliner as it can produce artifacts (especially at 3 T) due to electrical conductivity of the titanium oxide pigment.
   3. Ensure that the patient does not have any contraindications to perform MRI imaging⁹:
      1. Ask the patient about any metallic materials, which might include face masks, piercings, artificial limbs, magnetic dental implants, cerebral artery aneurysm clips.
      2. Ask the patient about metallic intraocular foreign bodies. For this, ask the patient if they have welded without proper protective gear.
      3. Ask the patient about any implantable devices might be incompatible with MRI, including pacemakers and insulin pumps, analgesic drugs, or chemotherapy pumps. In addition to this, cochlear implants/ear implant, Implantable neurostimulation systems, Implantable neurostimulation systems, Catheters with metallic components, are all contraindicated.
      4. Ask the patient about metallic foreign body left inside their body. This includes bullets, shotgun pellets, and metal shrapnel
      5. Ask the patient about any surgical clips or wire sutures, joint replacement or prosthesis, inferior vena cava (IVC) filter, ocular prosthesis, stents, or intrauterine device.
      6. Ask the patient if they have gotten a tattoo in the last 6 weeks.
      7. Ask the patient if they have undergone a colonoscopy procedure in the last eight weeks.
      8. Due to the confined space of the MRI machine, ask the patient if they have claustrophobia.
         NOTE: Difficulty might be found with patients having high body mass index (BMI).
   4. Explain to the patient that the exam is expected to take 15 minutes, where the patient needs to stay still.
2. After completing the instructions and making sure the patient fully comprehends the exam, obtain a signed consent.
3. During MRI image acquisition, lay the patient supine in the MRI machine, and fixate on a straight target during imaging without any head movement. For patients with poor visual acuity, use a sound stimulus to optimize fixation. More comprehensive methods for fixation involve shutting one eye, use of a fixation target centrally in the form of an LCD screen that changes colors, and use of ocular lubricants.
4. Ensure that the patient is aware that there is a squeeze button that can be pressed if they need anything while in the MRI machine. While a head coil can be used, an eye coil and an orbital coil may be more suited for ophthalmic imaging.
5. Input the following parameters for image acquisition: A T2-weighted fat suppression sequence (TR = 3000 milliseconds; TE = 90 milliseconds; TE = 100; field of view = 16 cm×16 cm; matrix = 296*384; slice thickness = 3 mm; slice gap = 0.3 mm). The final image analyzed was an oblique coronal image 3 mm behind the globe. It is important to note that while T2-weighted fat suppression sequence is generally used for optic nerve imaging, other sequences can be used, including T2 fast spin echo imaging.
6. Take a coronal cut of the optic nerve orthogonal (i.e., perpendicular) to the nerve 3 mm posterior to the globe. Use scout images in the transversal and oblique sagittal planes to ensure optimal optic nerve direction and optic nerve-globe junction positioning.
7. Assess the quality of gaze fixation by CSF distribution around the optic nerve, where it should be uniformly distributed around the optic nerve with almost equal thickness at all sides.
8. Repeat the process to image the optic nerve for the other side.

2. Image analysis

1. Download Fiji image processing package from (https://imagej.net/Fiji).
2. Upload the coronal image of optic nerve to ImageJ Fiji software for analysis by clicking File from the menu bar, followed by Open button. Choose the coronal image to be processed. Transfer the images to Fiji software without losing image quality during transfer, as image quality loss will lead to unreliable image analysis results.
3. Standardize the scale by specifying the number of pixels per a unit of length by drawing a straight line on the map scale. Then choose Set Scale from the Analyze menu bar. Specify the length of the line as appears on the map scale with the proper unite of length (i.e., mostly mm).
4. Convert the image into a grayscale using the image menu, and then choosing Type and 8-bit.
5. Quantify the range of intensity of white matter pixels.
   1. Using the Lasso selection tool (Plugin | Segmentation | Lasso tool), select a sufficient area of white matter, making sure not to include gray matter area during selection. We found that a total selected white matter area of around 1000 pixels is enough. Use Analyze and Measure tool to quantify the selected area.
6. Show the Histogram tool from Analyze menu, which shows the distribution of pixels intensity in the white matter area selected. Click on the Live box to make sure the histogram assesses the selected area. The graph on the histogram should show a normal distribution of intensity.
7. Calculate white matter intensity range as follows:
   Lower limit = mean intensity - (3* standard deviation)
   Upper limit = mean intensity + (3* standard deviation)
8. Open the Threshold tool from Image menu, followed by the Adjust function. Specify the range calculated from the previous step. Tick only dark background function and specify black and white annotation B&W from drop list, then click apply. The mask for white matter present within the optic disc will appear.
9. Using the Lasso selection tool (Plugin | Segmentation | Lasso tool), select the black area representing the optic disc.
10. Use the Measure function from the Analyze menu bar, which will calculate the area marked by the threshold function in mm.

Results

The cup to disc ratio for a 30-year-old male patient presenting for a checkup ophthalmology exam was 0.8 (Figure 1A), which is suspicious and might be suggestive of glaucoma. Upon performing an optical coherence tomography for nerve fiber layer thickness, we found that the nerve thickness was within the normal limits for age (Figure 1B). The patient was scheduled for an orbit MRI, where a coronal cut for optic nerve assessment was ordered and performed as per th...

Discussion

We described a protocol to assess and quantify optic nerve white matter that might be used for glaucoma patient assessment. The protocol uses widely available imaging sequences for image acquisition, and it uses the open-source Fiji software for image analysis. We standardized the image parameters that were previously found to be most accurate and highly reproducible in optic nerve image acquisition, including asking the patient to fixate straight ahead, using T2 with fat suppression sequence, and capturing the cross-sec...

Materials

Name	Company	Catalog Number	Comments
Magnetic resonance imaging (MRI) machine	Siemens Magnetom Verio	N/A	3T MRI scanner