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>

<ol>
	<li>Quigley, H. A., Broman, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+number+of+people+with+glaucoma+worldwide+in+2010+and+2020.">The number of people with glaucoma worldwide in 2010 and 2020.</a> The British Journal of Ophthalmology. 90 (3), 262-267 (2006).</li><li>Weinreb, R. N., Aung, T., Medeiros, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+and+treatment+of+glaucoma:+a+review.">The pathophysiology and treatment of glaucoma: a review.</a> JAMA. 311 (18), 1901-1911 (2014).</li><li>. Overview | Glaucoma: diagnosis and management | Guidance | NICE Available from: <a target="_blank" href="https://www.nice.org.uk/guidance/ng81">https://www.nice.org.uk/guidance/ng81</a> (2021)</li><li>Michelessi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+nerve+head+and+fibre+layer+imaging+for+diagnosing+glaucoma.">Optic nerve head and fibre layer imaging for diagnosing glaucoma.</a> The Cochrane Database of Systematic Reviews. (11), 008803 (2015).</li><li>Ramli, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+use+of+3T+MRI+in+assessment+of+optic+nerve+volume+in+glaucoma.">Novel use of 3T MRI in assessment of optic nerve volume in glaucoma.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 252 (6), 995-1000 (2014).</li><li>AlRyalat, S. A., Muhtaseb, R., Alshammari, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulating+a+colour-blind+ophthalmologist+for+diagnosing+and+staging+diabetic+retinopathy.">Simulating a colour-blind ophthalmologist for diagnosing and staging diabetic retinopathy.</a> Eye. , 1-4 (2020).</li><li>Chang, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+Nerve+Diffusion+Tensor+Imaging+Parameters+and+Their+Correlation+With+Optic+Disc+Topography+and+Disease+Severity+in+Adult+Glaucoma+Patients+and+Controls.">Optic Nerve Diffusion Tensor Imaging Parameters and Their Correlation With Optic Disc Topography and Disease Severity in Adult Glaucoma Patients and Controls.</a> Journal of Glaucoma. 23 (8), 513-520 (2014).</li><li>Omodaka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+magnetic+resonance+imaging+optic+nerve+parameters+to+optical+coherence+tomography+and+the+visual+field+in+glaucoma.">Correlation of magnetic resonance imaging optic nerve parameters to optical coherence tomography and the visual field in glaucoma.</a> Clinical &amp; Experimental Ophthalmology. 42 (4), 360-368 (2014).</li><li>Ghadimi, M., Sapra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+Resonance+Imaging+Contraindications.">Magnetic Resonance Imaging Contraindications.</a> StatPearls. , (2021).</li><li>B&#228;uerle, J., Schuchardt, F., Schroeder, L., Egger, K., Weigel, M., Harloff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+and+accuracy+of+optic+nerve+sheath+diameter+assessment+using+ultrasound+compared+to+magnetic+resonance+imaging.">Reproducibility and accuracy of optic nerve sheath diameter assessment using ultrasound compared to magnetic resonance imaging.</a> BMC Neurology. 13 (1), 187 (2013).</li><li>Wang, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orbital+Cerebrospinal+Fluid+Space+in+Glaucoma:+The+Beijing+Intracranial+and+Intraocular+Pressure+(iCOP)+Study.">Orbital Cerebrospinal Fluid Space in Glaucoma: The Beijing Intracranial and Intraocular Pressure (iCOP) Study.</a> Ophthalmology. 119 (10), 2065-2073 (2012).</li><li>Weigel, M., Lagr&#232;ze, W. A., Lazzaro, A., Hennig, J., Bley, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+Quantitative+High-Resolution+Magnetic+Resonance+Imaging+of+the+Optic+Nerve+at+3.0+Tesla.">Fast and Quantitative High-Resolution Magnetic Resonance Imaging of the Optic Nerve at 3.0 Tesla.</a> Investigative Radiology. 41 (2), 83-86 (2006).</li><li>Yiannakas, M. C., Toosy, A. T., Raftopoulos, R. E., Kapoor, R., Miller, D. H., Wheeler-Kingshott, C. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRI+Acquisition+and+Analysis+Protocol+for+In+Vivo+Intraorbital+Optic+Nerve+Segmentation+at+3T.">MRI Acquisition and Analysis Protocol for In Vivo Intraorbital Optic Nerve Segmentation at 3T.</a> Investigative Ophthalmology &amp; Visual Science. 54 (6), 4235-4240 (2013).</li><li>Al-Haddad, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+Nerve+Measurement+on+MRI+in+the+Pediatric+Population:+Normative+Values+and+Correlations.">Optic Nerve Measurement on MRI in the Pediatric Population: Normative Values and Correlations.</a> American Journal of Neuroradiology. 39 (2), 369-374 (2018).</li><li>Mncube, S. S., Goodier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+measurements+of+the+optic+nerve,+optic+nerve+sheath+and+optic+chiasm+in+the+adult+population.">Normal measurements of the optic nerve, optic nerve sheath and optic chiasm in the adult population.</a> South African Journal of Radiology. 23 (1), 7 (2019).</li><li>Nguyen, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-High+Field+Magnetic+Resonance+Imaging+of+the+Retrobulbar+Optic+Nerve,+Subarachnoid+Space,+and+Optic+Nerve+Sheath+in+Emmetropic+and+Myopic+Eyes.">Ultra-High Field Magnetic Resonance Imaging of the Retrobulbar Optic Nerve, Subarachnoid Space, and Optic Nerve Sheath in Emmetropic and Myopic Eyes.</a> Translational Vision Science &amp; Technology. 10 (2), (2021).</li><li>Lagr&#232;ze, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrobulbar+Optic+Nerve+Diameter+Measured+by+High-Speed+Magnetic+Resonance+Imaging+as+a+Biomarker+for+Axonal+Loss+in+Glaucomatous+Optic+Atrophy.">Retrobulbar Optic Nerve Diameter Measured by High-Speed Magnetic Resonance Imaging as a Biomarker for Axonal Loss in Glaucomatous Optic Atrophy.</a> Investigative Ophthalmology &amp; Visual Science. 50 (9), 4223-4228 (2009).</li><li>Nielsen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+Resonance+Imaging+at+3.0+Tesla+Detects+More+Lesions+in+Acute+Optic+Neuritis+Than+at+1.5+Tesla.">Magnetic Resonance Imaging at 3.0 Tesla Detects More Lesions in Acute Optic Neuritis Than at 1.5 Tesla.</a> Investigative Radiology. 41 (2), 76-82 (2006).</li><li>Mafee, M. F., Rapoport, M., Karimi, A., Ansari, S. A., Shah, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orbital+and+ocular+imaging+using+3-+and+1.5-T+MR+imaging+systems.">Orbital and ocular imaging using 3- and 1.5-T MR imaging systems.</a> Neuroimaging Clinics of North America. 15 (1), 1-21 (2005).</li><li>Gala, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+optic+nerve.">Magnetic resonance imaging of optic nerve.</a> The Indian Journal of Radiology &amp; Imaging. 25 (4), 421-438 (2015).</li><li>Gao, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+Nerve+Cross-Sectional+Area+Measurement+with+High-Resolution,+Isotropic+MRI+in+Optic+Neuritis+(P6.159).">Optic Nerve Cross-Sectional Area Measurement with High-Resolution, Isotropic MRI in Optic Neuritis (P6.159).</a> Neurology. 84 (14), (2015).</li><li>Zou, H., M&#252;ller, H. J., Shi, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-spatial+sounds+regulate+eye+movements+and+enhance+visual+search.">Non-spatial sounds regulate eye movements and enhance visual search.</a> Journal of Vision. 12 (5), 2 (2012).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Connective+Tissue+Components+of+Optic+Nerve+Head+Cupping+in+Monkey+Experimental+Glaucoma+Part+1:+Global+Change.">The Connective Tissue Components of Optic Nerve Head Cupping in Monkey Experimental Glaucoma Part 1: Global Change.</a> Investigative Ophthalmology &amp; Visual Science. 56 (13), 7661-7678 (2015).</li><li>Mwanza, J. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+nerve+fibre+layer+thickness+floor+and+corresponding+functional+loss+in+glaucoma.">Retinal nerve fibre layer thickness floor and corresponding functional loss in glaucoma.</a> The British Journal of Ophthalmology. 99 (6), 732-737 (2015).</li></ol>

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-sec...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 th...

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

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis

We evaluated a magnetic resonance venography (MRV) approach with gadofosveset to quantify total thrombus volume changes as the principal criterion for treatment efficacy&#160;in a multicenter randomized study comparing edoxaban monotherapy with a heparin/warfarin regimen for acute, symptomatic lower extremities deep vein thrombosis (DVT) treatment. We also used a direct thrombus imaging approach (DTHI, without the use of a contrast agent) to quantify fresh thrombus. We then sought to evaluate the reproducibility of the analysis methodology and applicability of using 3D magnetic resonance venography and direct thrombus imaging for the quantification of DVT in a multicenter trial setting. From 10 randomly selected subjects participating in the edoxaban Thrombus Reduction Imaging Study (eTRIS), total thrombus volume in the entire lower extremity deep venous system was quantified bilaterally. Subjects were imaged using 3D-T1W gradient echo sequences before (direct thrombus imaging, DTHI) and 5 min after injection of 0.03 mmol/kg of gadofosveset trisodium (magnetic resonance venography, MRV). The margins of the DVT on corresponding axial, curved multi-planar reformatted images were manually delineated by two observers to obtain volumetric measurements of the venous thrombi. MRV was used to compute total DVT volume, whereas DTHI was used to compute volume of fresh thrombus. Intra-class correlation (ICC) and Bland Altman analysis were performed to compare inter and intra-observer variability of the analysis. The ICC for inter and intra-observer variability was excellent (0.99 and 0.98, p &#60;0.001, respectively) with no bias on Bland-Altman analysis for MRV images. For DTHI images, the results were slightly lower (ICC = 0.88 and 0.95 respectively, p &#60;0.001), with bias for inter-observer results on Bland-Altman plots. This study showed feasibility of thrombus volume estimation in DVT using MRV with gadofosveset trisodium, with good intra- and inter-observer reproducibility in a multicenter setting.

The goal of this study is to use magnetic resonance venography with long-circulating gadolinium-based contrast agent and direct thrombus imaging for quantitative evaluation of DVT volume in a multicenter, clinical trial setting. Inter- and intra-observer variability assessments were conducted, and reproducibility of the protocol was determined.

We evaluated a magnetic resonance venography (MRV) approach with gadofosveset to quantify total thrombus volume changes as the principal criterion for ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

Nerve Ultrasound Protocol to Detect Dysimmune Neuropathies

Nerve ultrasound is increasingly used in the differential diagnosis of polyneuropathy as a complementary tool to nerve conduction studies. Morphological alterations of the peripheral nerves, such as increasing the cross-sectional area (CSA), have been described in various immune-mediated polyneuropathies. The most prominent morphological changes in nerve ultrasound have been described for the chronic inflammatory demyelinating polyneuropathy (CIDP)-spectrum disease. CIDP may be distinguished from hereditary and other polyneuropathies by measuring the extent and pattern of nerve swellings (CSA increase). Typical findings in demyelinating inflammatory neuropathies are multifocal nerve swellings with inhomogeneous fascicular structure, while CSA increase in demyelinating hereditary neuropathies occurs in a more generalized and homogenous manner. In other non-inflammatory axonal neuropathies, nerves can appear with normal or slight CSA increases, especially in typical entrapment sites. This article presents technical requirements for nerve ultrasound, an examination procedure using a standardized examination protocol, current reference values for the CSA, and typical sonographic pathological findings in patients with inflammatory neuropathies.

This article presents a protocol for nerve ultrasound in polyneuropathies to aid the diagnosis of inflammatory neuropathies.

Nerve ultrasound is increasingly used in the differential diagnosis of polyneuropathy as a complementary tool to nerve conduction studies. Morphological ...

Preventing Posterior Capsular Opacification Through IOL Rotation in Cataract Surgeries

Rotating the Intraocular Lens to Prevent Posterior Capsular Opacification in Cataract Surgeries

Posterior capsule opacification (PCO) is a common postoperative complication of extracapsular cataract surgery, which is caused by the proliferation and migration of lens epithelial cells and can affect long-term visual outcomes significantly. The most effective treatment for PCO is neodymium-doped yttrium aluminum garnet (Nd:YAG) laser capsulotomy; however, this treatment is associated with posterior segment complication and can break the stability of capsular bag, affecting the position and function of trifocal or toric intraocular lenses (IOLs). Advances in surgical procedures, IOL design, and pharmacy have reduced the rate of PCO in recent years, concentrating on the inhibition of proliferative lens epithelial cells (LECs). This protocol aimed to clear LECs more thoroughly during phacoemulsification and IOL implantation. The first several steps, including clear corneal incision, continuous circular capsulorhexis, hydrodissection, hydrodelineation, and phacoemulsification, were completed as conventional procedures. After placing the IOL into the capsular bag, rotation of the IOL by at least 360&#176; was performed using an irrigation/aspiration tip or a hook, with slight stress on the posterior capsule. Some residuals occurred in the originally transparent capsular bag after rotation of the IOLs. Then, these materials and the viscoelastic were cleared completely using an irrigation/aspiration system. A clear posterior capsule was observed after the surgery in patients undergoing this method. This method of rotating IOLs is a simple, effective, and safe way to prevent PCO by clearing residual LECs and can be carried out without extra tools or skills.

Author Spotlight: Enhancing Visual Outcomes in Cataract Surgery: A Novel Technique to Prevent Posterior Capsular Opacification Through IOL Rotation 

The present protocol describes removing residual epithelial cells by rotating the intraocular lens in extracapsular cataract surgery without extra tools for preventing posterior capsular opacification.

Posterior capsule opacification (PCO) is a common postoperative complication of extracapsular cataract surgery, which is caused by the proliferation and ...

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 ...

RPNI Surgery for Treating Postamputation Pain

Regenerative Peripheral Nerve Interface: Surgical Protocol for a Randomized Controlled Trial in Postamputation Pain

Surgical procedures, including nerve reconstruction and end-organ muscle reinnervation, have become more prominent in the prosthetic field over the past decade. Primarily developed to increase the functionality of prosthetic limbs, these surgical procedures have also been found to reduce postamputation neuropathic pain. Today, some of these procedures are performed more frequently for the management and prevention of postamputation pain than for prosthetic fitting, indicating a significant need for effective solutions to postamputation pain. One notable emerging procedure in this context is the Regenerative Peripheral Nerve Interface (RPNI). RPNI surgery involves an operative approach that entails splitting the nerve end longitudinally into its main fascicles and implanting these fascicles within&#160;free denervated and devascularized muscle grafts. The RPNI procedure takes a proactive stance in addressing freshly cut nerve endings, facilitating painful neuroma prevention and treatment by enabling the&#160;nerve to regenerate and innervate an end organ, i.e., the free muscle graft. Retrospective studies have shown RPNI&#39;s effectiveness in alleviating postamputation pain and preventing the formation of painful neuromas. The increasing frequency of utilization of this approach has also given rise to variations in the technique. This article aims to provide a step-by-step description of the RPNI procedure, which will serve as the standardized procedure employed in an international, randomized controlled trial (ClinicalTrials.gov, NCT05009394). In this trial, RPNI is compared to two other surgical procedures for postamputation pain management, specifically, Targeted Muscle Reinnervation (TMR) and neuroma excision coupled with intra-muscular transposition and burying.

Author Spotlight: Regenerative Peripheral Nerve Interface (RPNI) Surgery in Postamputation Pain Management

Here, we describe the surgical procedure to perform Regenerative Peripheral Nerve Interface (RPNI) surgery for treating postamputation neuropathic pain in the context of an international, randomized controlled trial (RCT) (ClinicalTrials.gov, NCT05009394). The RCT compares RPNI with two other surgical techniques, namely, Targeted Muscle Reinnervation (TMR) and neuroma excision combined with intra-muscular transposition.

Surgical procedures, including nerve reconstruction and end-organ muscle reinnervation, have become more prominent in the prosthetic field over the past ...