Name: quantification of optic nerve cross sectional area on mri: a novel protocol using fiji software
Uploaded: 2021-09-04T15:00:00.000+00:00
Duration: 8 min 57 s
Description: Watch this Scientific Journal Video about Quantification of Optic Nerve Cross Sectional Area on MRI: A Novel Protocol using Fiji Software at JoVE.com

We would like to thank Faris Haddad and Hasan El-Isa for their important contribution in video filming and development.

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).
<ol>
	<li>Fully explain the examination to the patient. The following include instructions and explanations that need to be mentioned to the patient.
	<ol>
		<li>Explain to the patient that they will need to change clothes and wear a special gown for imaging.</li>
		<li>Have patients remove any worn eyeliner as it can produce artifacts (especially at 3 T) due to electrical conductivity of the titanium oxide pigment.</li>
		<li>Ensure that the patient does not have any contraindications to perform MRI imaging9:
		<ol>
			<li>Ask the patient about any metallic materials, which might include face masks, piercings, artificial limbs, magnetic dental implants, cerebral artery aneurysm clips.</li>
			<li>Ask the patient about metallic intraocular foreign bodies. For this, ask the patient if they have welded without proper protective gear.</li>
			<li>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.</li>
			<li>Ask the patient about metallic foreign body left inside their body. This includes bullets, shotgun pellets, and metal shrapnel</li>
			<li>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.</li>
			<li>Ask the patient if they have gotten a tattoo in the last 6 weeks.</li>
			<li>Ask the patient if they have undergone a colonoscopy procedure in the last eight weeks.</li>
			<li>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).</li>
		</ol>
		</li>
		<li>Explain to the patient that the exam is expected to take 15 minutes, where the patient needs to stay still.</li>
	</ol>
	</li>
	<li>After completing the instructions and making sure the patient fully comprehends the exam, obtain a signed consent.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#215;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Repeat the process to image the optic nerve for the other side.</li>
</ol>
2. Image analysis
<ol>
	<li>Download Fiji image processing package from (https://imagej.net/Fiji).</li>
	<li>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.</li>
	<li>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).</li>
	<li>Convert the image into a grayscale using the image menu, and then choosing Type and 8-bit.</li>
	<li>Quantify the range of intensity of white matter pixels.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Calculate white matter intensity range as follows: 
	Lower limit = mean intensity - (3* standard deviation) 
	Upper limit = mean intensity + (3* standard deviation)</li>
	<li>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&#38;W from drop list, then click apply. The mask for white matter present within the optic disc will appear.</li>
	<li>Using the Lasso selection tool (Plugin | Segmentation | Lasso tool), select the black area representing the optic disc.</li>
	<li>Use the Measure function from the Analyze menu bar, which will calculate the area marked by the threshold function in mm.</li>
</ol>

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All authors declare no conflict of interest.

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-sectional area 3 mm behind the globe. In addition, we described a detailed image analysis method that eliminates manual segmentation and corrects for signal variability between patients. The importance of this protocol is that it eliminates the variation in region of interest (ROI) segmentation by the radiologist, which is usually the main source of error in optic nerve assessment on MRI12. While we tried to provide normative data for optic nerve cross sectional area with Table 1, further data using the standard protocol described is needed for the use and comparison in clinical settings. Such data need to accommodate different age groups due to the age variation in optic nerve size, as shown in Table 1. Such variation is not evident between genders15, but was recently suggested to be present for refractive error16.
Previous studies applied different methodologies for optic nerve white matter quantification, and they mostly used the software present in their workstation for image analysis. Initial studies on optic nerve assessment adopted a quantification approach based on cross sectional area, using manual segmentation by technicians or radiologists12,17. Wang et al. also used manual segmentation of optic nerve cross-sectional area at different distances from the globe for correlation with OCT 11. Omodaka et al. used the average cross-sectional area on the coronal cut and the length of the optic nerve from the disc to the optic chiasm on the axial cut through manual annotation to extract indicators of the optic nerve for correlation with OCT8. Despite being correlated with OCT, reproducibility of this method might not yield the required accuracy for longitudinal assessment of optic nerve. Ramli et al. quantified the volume of the optic nerve through manual segmentation of the isointense signal at all axial sections 5, an approach that might miss the optic nerve matter not captured by the axial sections themselves, human error during image manual segmentation, or even in the determination of the optic nerve length to be included in the quantification assessment.
While different studies used the assessment of the cross-sectional area of the optic nerve, they differed in the distance of measurements from globe. Wang et al. assessed 3 mm, 9 mm, and 15 mm behind the globe, and found that the 3 mm cross-sectional assessment had the highest correlation with intraocular pressure11. B&#228;uerle et al. analyzed the reproducibility of assessment of the optic nerve on MRI at 3 mm and 5 mm behind the globe, and they found a good assessment for both cases10. Lagr&#232;ze et al. measured the cross-sectional area 5 mm, 10 mm, and 15 mm behind the globe and found that the cross-sectional assessment was most accurate in the 5 mm cross sectional area compared to measurements further from the globe17. In this protocol, we used a 3 T MRI for images acquisition, where its use in optic nerve assessment was previously found to be superior to 1.5 T MRI18,19. The increasingly utilized 7 T MRI might also provide superior results but will also require its normative values. Regarding MRI sequence used, we used T2 fat suppression sequence, mainly due to its wide availability and its inherent ability to delineate the optic nerve surrounding CSF after eliminating the surrounding intraconal fat. Previous studies used other sequences with reliable results, including the half-Fourier single-shot turbo spin-echo (HASTE) sequence and Diffusion Tensor Imaging (DTI) sequence7,12, which might not be widely available.
An important aspect to consider during image acquisition is making sure that the patient is fixating on a straight target, as fixating on a non-straight target during imaging will yield a non-accurate optic nerve quantification12. Fixation in OCT is monocular on a close target, requiring the patient to have good visual acuity in the eye to be assessed to see the near target with one eye, whereas for MRI the target is further away, the fixation is binocular, and requires fewer visual demands. However, fixation might still be an issue for patients who have high refractive error or poor vision. While the use of MRI to assess and follow patients with glaucoma might not be feasible in the presence of low cost, simpler imaging techniques, including the OCT, MRI can be useful in special situations where OCT do not provide conclusive data, or OCT itself cannot be obtained, such as in the presence of significant ocular medial opacity. Furthermore, the described protocol can be used when MRI imaging is warranted in cases of unexplained optic neuropathy to exclude secondary causes20,21.
One of the main limitations of this protocol is the inability to assess patients who cannot properly fixate, including patients with poor visual acuity in both eyes. In this regard, the use of sound stimulus will improve the quality of fixation during image acquisition22. Moreover, as a new methodology, future studies are needed to depict normal values for MRI-based cross-sectional areas for optic nerve white matter. The importance of establishing normal values is further stressed by the fact that optic nerve is also composed of significant amount of connective tissue23, a tissue that does not have similar functional abilities as nerve fibers. While quantification of optic nerve fiber layer thickness in OCT might provide a false impression of remanent nerve tissue due to the inclusion of connective tissue in the quantification process24, such a false impression is not present in this MRI-based quantification method. Motion artifacts might also lead to blur in images, especially upon eye movement during exam. While it should be avoided during imaging, establishing white matter range will reduce the impact of such artifacts on the accuracy of optic nerve white matter quantification, as the changes caused by the motion artifact on cerebral white matter is almost like the optic nerve&#39;s white matter.
The main strength of the current protocol is the elimination of inter-individual differences during optic nerve quantification, even when performed by non-specialized medical doctors or technicians. In addition, it used a widely available open-source software for image analysis. While it is not feasible to do a dedicated MRI imaging for optic nerve quantification, especially in the presence of the OCT, it is recommended to perform this protocol during MRI imaging done for other purposes, including the exclusion of secondary causes of optic neuropathy and glaucoma.

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 MRI7,8. 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&#39;s Fiji software for image processing and quantification.

<table><tbody><tr><td>Magnetic resonance imaging (MRI) machine</td><td>Siemens Magnetom Verio</td><td>N/A</td><td>3T MRI scanner</td></tr></tbody></table>

quantification of optic nerve cross sectional area on mri: a novel protocol using fiji software

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.

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 the abovementioned protocol.
We obtained a coronal MRI cut, 3 mm behind the optic disc. The white matter mean intensity was 94.372 (SD 7.085), which resulted in white matter intensity range of:
Lower limit = 94.372 - 21.255 = 73.117
Upper limit = 94.372 + 21.255 = 115.627
Figure 2&#160;shows the coronal image (Figure 2A), the coronal image after applying the white matter threshold using the calculated upper and lower limits (Figure 2B), and the optic nerve white matter for quantification (Figure 2C). The cross-sectional area for the white matter of the left optic nerve was 6.9 mm2 (0.069 cm2), which is within the normal limits for his age, as shown in Table 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62752/62752fig01.jpg" /> 
Figure 1: Fundus image showing high cup to disc ratio, which might be suggestive of glaucoma (A). An optical coherence tomography for the nerve fiber layer (NFL) showing a within normal limits NFL (B). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62752/62752fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62752/62752fig02.jpg" /> 
Figure 2: Coronal T2 -weighted fat suppressed MRI image obtained perpendicular to the optic nerve 3 mm behind the optic disc (A). The same coronal cut after applying a pre-calculated threshold range (B). Optic nerve white matter (C). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62752/62752fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Study</td>
			<td>Sample size</td>
			<td>Age (Years)</td>
			<td>Mean cross sectional area (mm2)</td>
			<td>Imaging sequence</td>
		</tr>
		<tr>
			<td>B&#228;uerle, 2013. 10</td>
			<td>15</td>
			<td>Mean (SD) 24.5&#8201;&#177;&#8201;0.8</td>
			<td>5.69&#8201;&#177;&#8201;0.77</td>
			<td>T2-weighted turbo spin echo (TSE) sequence</td>
		</tr>
		<tr>
			<td>Wang 2012. 11</td>
			<td>21</td>
			<td>Mean (SD) 51.6&#177;12.0</td>
			<td>5.03 &#177; 0.35</td>
			<td>T2-weighted fast recovery fast spin echo (FRFSE) sequence</td>
		</tr>
		<tr>
			<td>Weigel, 2006. 12</td>
			<td>32</td>
			<td>Mean (range) 25 (22&#8211;39)</td>
			<td>5.7 &#177; 0.6</td>
			<td>T2-weighted turbo spin echo (TSE) sequence</td>
		</tr>
		<tr>
			<td>Yiannakas, 2013. 13</td>
			<td>8</td>
			<td>Mean (range) 31 (29&#8211;33)</td>
			<td>6.2 (1.3)</td>
			<td>T2-fat-suppressed</td>
		</tr>
		<tr>
			<td>Al-Haddad, 2018. 14</td>
			<td>211</td>
			<td>Median (interquartile) 8.6 (3.9&#8211;13.3)</td>
			<td>4.0 &#177; 0.20 *</td>
			<td>T1-weighted inversion recovery sequence</td>
		</tr>
		<tr>
			<td colspan="5">*Calculated using the provided optic nerve diameter.</td>
		</tr>
	</tbody>
</table>
Table 1 shows the normal range of optic nerve cross sectional diameter using MRI 3mm from globe, as found by previous studies.

Watch this Scientific Journal Video about Quantification of Optic Nerve Cross Sectional Area on MRI: A Novel Protocol using Fiji Software at JoVE.com

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

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.

Optic nerve assessment is an important aspect of glaucoma diagnosis and follow-up. This project describes a protocol for a unified methodology of optic ...

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3.9K Views.  The University of Jordan. 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.

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

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Neuroscience

Measuring Connectivity in the Primary Visual Pathway in Human Albinism Using Diffusion Tensor Imaging and Tractography

In albinism, the number of ipsilaterally projecting retinal ganglion cells (RGCs) is significantly reduced. The retina and optic chiasm have been proposed as candidate sites for misrouting. Since a correlation between the number of lateral geniculate nucleus (LGN) relay neurons and LGN size has been shown, and based on previously reported reductions in LGN volumes in human albinism, we suggest that fiber projections from LGN to the primary visual cortex (V1) are also reduced. Studying structural differences in the visual system of albinism can improve the understanding of the mechanism of misrouting and subsequent clinical applications. Diffusion data and tractography are useful for mapping the OR (optic radiation). This manuscript describes two algorithms for OR reconstruction in order to compare brain connectivity in albinism and controls.An MRI scanner with a 32-channel head coil was used to acquire structural scans. A T1-weighted 3D-MPRAGE sequence with 1 mm3 isotropic voxel size was used to generate high-resolution images for V1 segmentation. Multiple proton density (PD) weighted images were acquired coronally for right and left LGN localization. Diffusion tensor imaging (DTI) scans were acquired with 64 diffusion directions. Both deterministic and probabilistic tracking methods were run and compared, with LGN as the seed mask and V1 as the target mask. Though DTI provides relatively poor spatial resolution, and accurate delineation of OR may be challenging due to its low fiber density, tractography has been shown to be advantageous both in research and clinically. Tract based spatial statistics (TBSS) revealed areas of significantly reduced white matter integrity within the OR in patients with albinism compared to controls. Pairwise comparisons revealed a significant reduction in LGN to V1 connectivity in albinism compared to controls. Comparing both tracking algorithms revealed common findings, strengthening the reliability of the technique.

This manuscript describes deterministic and probabilistic algorithms for white matter (WM) reconstruction, used to examine differences in optic radiation (OR) connectivity between albinism and controls. Although probabilistic tractography follows the true course of nerve fibers more closely, deterministic tractography was run to compare the reliability and reproducibility of both techniques.

In albinism, the number of ipsilaterally projecting retinal ganglion cells (RGCs) is significantly reduced. The retina and optic chiasm have been proposed ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Bioengineering

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Meni&#232;re's Disease Ears and Normal Hearing Ears

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this patient group but has yet to be confirmed. This protocol describes a method of in vivo evaluation of neural changes especially well suitable for cranial nerve analysis using magnetic resonance imaging (MRI). MD-patients and normal hearing persons were examined in a 3-T MR-scanner using a scan protocol including strongly T2-weighted 3D gradient-echo-sequence (3D-CISS). In the patient group, MD was additionally confirmed using MRI-based assessment of endolymphatic hydrops. Morphometric analysis was performed using a freeware DICOM viewer. Evaluation of cranial nerves included measurements of cross-sectional areas (CSAs) of the nerves at different levels as well as orthogonal diametric measurements.

To evaluate morphological changes of cranial nerves such as loss of neural structures or swelling of cranial nerves in Meni&#232;re&#39;s Disease (MD) or in healthy persons in vivo, a protocol of evaluation has been developed using magnetic resonance imaging (MRI). Additional MRI-based confirmation of MD was performed.

Analysis of neural structures in Meni&#232;re's Disease (MD) is of importance, since a loss of such structures has previously been proposed for this ...

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of the retina. This process, referred to as retinal vascular reactivity (RVR), is mediated by neurovascular coupling, which is impaired very early in retinal vascular diseases such as diabetic retinopathy. Therefore, a clinically feasible method of assessing vascular function may be of significant interest in both research and clinical settings. Recently, in vivo imaging of the retinal vasculature at the capillary level has been made possible by the FDA approval of optical coherence tomography angiography (OCTA), a noninvasive, minimal risk and dyeless angiography method with capillary level resolution. Concurrently, physiological and pathological changes in RVR have been shown by several investigators. The method shown in this manuscript is designed to investigate RVR using OCTA with no need for alterations to the clinical imaging procedures or device. It demonstrates real time imaging of the retina and retinal vasculature during exposure to hypercapnic or hyperoxic conditions. The exam is easily performed with two personnel in under 30 min with minimal subject discomfort or risk. This method is adaptable to other ophthalmic imaging devices and the applications may vary based on the composition of the gas mixture and patient population. A strength of this method is that it allows for an investigation of retinal vascular function at the capillary level in human subjects in vivo. Limitations of this method are largely those of OCTA and other retinal imaging methods including imaging artifacts and a restricted dynamic range. The results obtained from the method are OCT and OCTA images of the retina. These images are amenable to any analysis that is possible on commercially available OCT or OCTA devices. The general method, however, can be adapted to any form of ophthalmic imaging.

This article describes a method for measuring retinal vasculature reactivity in vivo with human subjects using a gas breathing provocation technique to deliver vasoactive stimuli while acquiring retinal images.

The vascular supply to the retina has been shown to dynamically adapt through vasoconstriction and vasodilation to accommodate the metabolic demands of ...

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Advancing Visual Cortex Mapping for Enhanced Evaluation of Vision Disorders

Functional Magnetic Resonance Imaging (fMRI) of the Visual Cortex with Wide-View Retinotopic Stimulation

High-resolution retinotopic blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) with a wide-view presentation can be used to functionally map the peripheral and central visual cortex. This method for measuring functional changes of the visual brain allows for functional mapping of the occipital lobe, stimulating &gt;100&#176; (&plusmn;50&#176;) or more of the visual field, compared to standard fMRI visual presentation setups which usually cover &lt;30&#176; of the visual field. A simple wide-view stimulation system for BOLD fMRI can be set up using common MR-compatible projectors by placing a large mirror or screen close to the subject's face and using only the posterior half of a standard head coil to provide a wide-viewing angle without obstructing their vision. The wide-view retinotopic fMRI map can then be imaged using various retinotopic stimulation paradigms, and the data can be analyzed to determine the functional activity of visual cortical regions corresponding to central and peripheral vision. This method provides a practical, easy-to-implement visual presentation system that can be used to evaluate changes in the peripheral and central visual cortex due to eye diseases such as glaucoma and the vision loss that may accompany them.

Author Spotlight: Insights into Visual Cortex Research Through Wide-View fMRI Mapping 

We have developed techniques for mapping the visual cortex function utilizing more of the visual field than is commonly used. This approach has the potential to enhance the evaluation of vision disorders and eye diseases.

High-resolution retinotopic blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) with a wide-view presentation can be ...

Optic Nerve Sheath Point of Care Ultrasound: Image Acquisition

The goal of this protocol is to develop a standardized method for acquiring images of the optic nerve sheath and measuring the optic nerve sheath diameter (ONSD). Diagnostic ultrasound of the ONSD to detect intracranial hypertension has traditionally faced many problems because of methodologic discrepancies. Due to inconsistencies in the measuring techniques, the potential for ONSD to become a non-invasive bedside monitoring tool for ICP has been hampered. However, establishing a transparent, consistent methodology for measuring the ONSD would support its use as a valid and reliable method of identifying intracranial hypertension. This is important as it has both high sensitivity and specificity in acute care settings. This narrative review describes ONSD POCUS image acquisition, including patient positioning, transducer selection, probe placement, the acquisition sequence, and image optimization. Further, visual aids are provided to assist in real-time during image acquisition. This method should be considered for patients for whom there are concerns regarding intracranial hypertension but who do not have an intracranial monitor in place.

Point-of-care ultrasound (POCUS) of the optic nerve sheath diameter (ONSD) has been shown to be useful in identifying patients with increased intracranial pressure (ICP). However, the non-standardized technique for this POCUS has hampered its use. We present a standardized image acquisition protocol for use in the acute care setting.

The goal of this protocol is to develop a standardized method for acquiring images of the optic nerve sheath and measuring the optic nerve sheath diameter ...

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

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In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Measuring Connectivity in the Primary Visual Pathway in Human Albinism Using Diffusion Tensor Imaging and Tractography

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

In Vivo Morphometric Analysis of Human Cranial Nerves Using Magnetic Resonance Imaging in Menière's Disease Ears and Normal Hearing Ears

Retinal Vascular Reactivity as Assessed by Optical Coherence Tomography Angiography

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Functional Magnetic Resonance Imaging (fMRI) of the Visual Cortex with Wide-View Retinotopic Stimulation

Optic Nerve Sheath Point of Care Ultrasound: Image Acquisition