We would like to thank the radiology technicians and nurses&#8217;staff of the Neuroradiology Area, IRCCS Istituto delle Scienze Neurologiche di Bologna, and their Coordinator Dr. Maria Grazia Crepaldi, for their collaboration.

The protocol is following the Local Research Committee&#39;s ethical standards and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
1. Selection of the patients
<ol>
	<li>Adopt the following inclusion criteria: patients older than 18 years old, fully collaborating, presenting a tumor of the skull base, or pituitary-diencephalic region.</li>
	<li>Exclude patients with contraindication to MRI (i.e., a pacemaker or ferromagnetic material) or presenting with emergent clinical conditions (i.e., intracranial hypertension, the acute visual loss that requires immediate surgery), or pregnant women, or patients with mental illness, or those who explicitly refuse to participate in this protocol.</li>
</ol>
2. Preparation for the MRI exam
<ol>
	<li>Before the MRI exam, administer the safety form to exclude significant contraindication to the exam and contrast agent injection: no ferromagnetic materials in the body, evaluation of MRI devices, safe or conditional, no pacemaker, no eye contact lenses on.</li>
	<li>If the scanner used for the MRI acquisition is a high field (e.g., 3 T, see Table of Materials), consider any potential additional contraindications related, for example, to neurostimulation devices.</li>
	<li>Check whether the patient has claustrophobia.</li>
	<li>Ensure that the patient has read and signed the MRI consent form to acknowledge the imaging exam&#39;s risks and benefits.</li>
	<li>Have a neuropsychologist perform a general evaluation and a targeted neurocognitive assessment based on the tumor location.</li>
	<li>Administer the Edinburgh inventory to evaluate handedness dominance29.</li>
</ol>
3.&#160;Positioning of the patient in the scanner
<ol>
	<li>Give earplugs to the patient to reduce MRI noise.</li>
	<li>Head movements can affect imaging quality; thus, use foam pads to reduce head movements, immobilizing the head inside the MRI coil.</li>
	<li>Provide an emergency alarm button to the patient in case of need to interrupt the exam.</li>
	<li>Switch on the camera and microphone inside the scanner to monitor, speak, and listen to the patient from the MRI acquisition room outside the scanner.</li>
</ol>
4. Brain MRI protocol setting and acquisition parameters
<ol>
	<li>Acquire a standardized multimodal MRI protocol high-field scanner (1.5 T or 3T). The following sequence parameters refer to a 3 T MRI, using a head-neck high-density array coil (64 channels).</li>
	<li>Acquire high-resolution and volumetric anatomical sequences: T1-weighted pre- and post-gadolinium contrast agent administration and FLAIR T2-weighted.</li>
	<li>For T1 and T2 weighted images acquire continuous sagittal slices providing isotropic resolution of 1x1x1 mm3 scanning time of about 5 min per sequence.</li>
	<li>Acquire a high-resolution T2-weighted sequence and localize the tumor area for cranial nerve visualization: a volumetric CISS (Constructive Interference in Steady State) with voxel dimension of 0.5x0.5x0.5 mm3 (scanning time of about 9 minutes).</li>
	<li>Acquire diffusion-weighted sequences using single-shot echo-planar images (EPI), voxel dimension of 2x2x2 mm3, 64 magnetic gradient directions with b-value of 2000 s/mm2, echo time of 98 ms, and relaxation time of 4300 ms.</li>
	<li>Acquire five volumes with null b-value at the beginning of the diffusion-weighted acquisition with phase encoding direction set to anterior-posterior (for diffusion weighted images total scanning time of 5 minutes).</li>
	<li>Additionally, acquire three volumes with null b-value but reversed phase encoding direction, posterior-anterior, to correct imaging distortions due to the EPI acquisition (scanning time of 42 seconds). Continuous near-axial slices are acquired.</li>
	<li>Acquire additional sequences to investigate specific tumor features, such as multi- or single-voxel MRI-spectroscopy localized in the tumor area. 
	&#8203;NOTE: The total scanning time duration is about 30 minutes, excluding patient preparation for the MRI exam.</li>
</ol>
5. Brain MR images pre-processing
<ol>
	<li>Convert the MRI data from the imaging format adopted by MRI acquisition consoles, DICOM (.dcm), to the NIFTI format (.nii) used in advanced imaging analyses.</li>
	<li>Run the dcm2niix function (https://github.com/rordenlab/dcm2niix). Set as input files dicom images and as output the corresponding .nii files: T1.nii, Flair.nii, T1_contrast.nii, DTI_b2000.nii and DTI_b0_flip.nii.</li>
	<li>Install the FSL (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki) and MRtrix3 (https://www.mrtrix.org) software needed for the advanced imaging analyses.</li>
	<li>Register the Flair.nii and T1_contrast.nii to the T1.nii image by running the FSL-flirt function, which performs a linear image registration.</li>
	<li>Register the DTI_b2000.nii image to the T1.nii by running the FSL-epi_reg function, which takes into account EPI imaging distortion artifacts.</li>
	<li>Run the FSL-topup function to correct phase encoding direction artifacts presenting the DTI_b2000.nii image. Set the DTI_b0_flip.nii inverse phase encoding acquisition as the &#34;in_main&#34; input file.</li>
	<li>Run the MRtrix3-dwidenoise function for imaging denoising with a principal component noise modeling.</li>
	<li>To correct eddy current and signal drop-out artifact, run the FSL-eddy function, and for MRI coil-induced signal inhomogeneities, the MRtrix3-dwibias correct function.</li>
	<li>Run the FSL-bet function to remove the scalp signal presenting the T1.nii image and rename the output file by using the &#34;_brain&#34; suffix: T1_brain.nii.</li>
</ol>
6. Tumor segmentation
<ol>
	<li>Install the itk-snap software (http://www.itksnap.org)&#160;30.</li>
	<li>Once the itk-snap software is installed, press File - Open Main Image and select the T1.nii image, then press File - Add Another Imager and upload the Flair.nii and T1_contrast.nii images, setting the semi-transparent overlay option.</li>
	<li>Inspect the tumor in the T1.nii, Flair.nii, and T1_contrast.nii images. Choose the anatomical plane to follow when drawing the lesion, e.g., axial.</li>
	<li>Place the pointer in one axial slice to start. In the Main Toolbar, select the Polygon Inspector icon and start drawing tumor boundaries by using the Freehand Drawing Style - Smooth curve or Polygon.</li>
	<li>Once finished drawing the tumor perimeter, close the curve linking the first and last dots, press Accept, and continue drawing in the next slice. For large tumor lesions, to accelerate the drawing process, skip some axial slices (e.g., three), and draw the lesion perimeter in interleaved slices.</li>
	<li>At the end of the lesion perimeter drawing, select Tools - Interpolate Labels, set the Label to/with interpolate as the tumor lesion and the Interpolate along a single axis as the axis orientation followed in drawing the tumor boundaries.</li>
	<li>Select Segmentation - Save Segmentation Image and name the tumor segmentation as Tumor_mask.nii by selecting the Nifti format option to save.</li>
</ol>
7. Tractography analysis
<ol>
	<li>Run the FSL-dtifit function to model diffusivity and the different spatial directions and obtain the following diffusion tensor maps: FA.nii, MD.nii, and V1.nii. Evaluate these DTI maps to access abnormal diffusivity values that may occur in the presence of tumor edema or infiltration.</li>
	<li>Run the MRtrix3-tckgen function with the default setting &#34;ifod2&#34; to perform probabilistic tractography and reconstruct the white matter pathways by modeling crossing-fibers issues31.</li>
	<li>Adopt a seed-target approach by setting the &#34;-seed_image&#34; and &#34;-include&#34; options based on a priori anatomical knowledge.</li>
	<li>Manually draw regions of interest (ROIs) set as seed or target for tractography. Alternatively, use atlas-based ROIs. See Mormina et at.32 for the optic radiation tractography, Hales et al.33 for the optic chiasm and optic cranial nerves, and Testa et al.34 for the pyramidal tracts.</li>
	<li>Launch the FSL-fsleyes image viewer, select Open, and choose images to inspect visually.</li>
	<li>In the FSL- fsleyes viewer, go to Setting - Ortho View 1 and activate the Edit Mode tool.</li>
	<li>Click the FSL-fsleyes pencil icon and draw the tractography ROIs.</li>
	<li>Install the Freesurfer (https://surfer.nmr.mgh.harvard.edu) software.</li>
	<li>Run the Freesurfer-Recon-all function on the T1.nii image to obtain the automatic cortical region segmentation to use as tractography ROIs.</li>
	<li>Run the FSL-epi_regregistration function, setting as input image the T1.nii, and reference image the DTI_b2000.nii, save the registration output matrix (T1_onto_DTI.mat).</li>
	<li>Use the obtained T1_onto_DTI.mat matrix to register the segmented ROIs to the DTI_b2000.nii image.</li>
	<li>Run the tractography using the MRtrix3-tckgen function.</li>
	<li>Run the MRtrix3-tckmap function to convert the &#34;.tck&#34; streamlines tractography output in the &#34;-template FA.nii&#34; image.</li>
	<li>Run the FSL-flirt function to linearly register the T1.nii image to the MNI152_T1_2mm_brain.nii template.</li>
	<li>Save the output matrix as T1_onto_MNI.mat. Run the FSL-convert_xfm function setting the &#34;-concat&#34; option as T1_onto_MNI.mat and T1_onto_DTI.mat, save the output matrix as DTI_onto_MNI.mat.</li>
</ol>
8. Tractography: along-tract analysis
<ol>
	<li>For an accurate description of DTI parameters, use along-tract algorithms, such as the Matlab-based algorithm that models the surface tract geometry with the Laplacian operator properties35.</li>
	<li>Install the Matlab software (https://matlab.mathworks.com) and request the along-tract code to the developing authors35.</li>
	<li>Alternatively, use the MRtrix3-tcksample function for along-tract analysis as Matlab requires a license.</li>
</ol>
9. 3D-rendering visualization
<ol>
	<li>Install the Surf Ice software (https://www.nitrc.org/plugins/mwiki/index.php/surfice:MainPage).</li>
	<li>In the Surf Ice command panel, click on Advanced - Convert voxelwise to mesh, select the nifti image to convert, save the resulting .obj file.</li>
	<li>In the Surf Ice command panel, click File - Open, and select the .obj file to visualize the 3D volume rendering.</li>
</ol>
10. Preoperative clinical examinations
<ol>
	<li>Perform bio-humoral endocrinological assessment, consisting of prolactin, TSH, freeT4, ACTH, cortisol, GH, LH, FSH, and serum tests total testosterone/estradiol, respectively in men and women.</li>
	<li>Analyze the 24-hour urine volume and serum and urine osmolality and sodium levels to determine the presence of diabetes insipidus.</li>
	<li>Perform an ophthalmological evaluation, including visual acuity measurement, computerized visual field assessment, and retinal optical coherence tomography (OCT).</li>
	<li>Perform a neurological physical examination, with a collection of anamnestic information about weight gain, the sensation of hunger, continuously monitoring rectal temperature every 2 min for 24 h using a portable device to evaluate the circadian temperature rhythm, and 24 h sleep-wake cycle recording (including an electroencephalogram, right and left electro-oculogram, electrocardiogram, and electromyogram of mylohyoid and left and right tibialis muscles)36,37,38.</li>
</ol>
11. Surgical planning
<ol>
	<li>Discuss in a collegial team meeting each patient candidate to surgery, based on the results of tumor segmentation and relationship with the functional eloquent neural structures (optic nerves and chiasm, pituitary stalk, third ventricle, internal carotid artery, anterior cerebral artery-anterior communicating artery (ACA-ACoA) complex, basilar artery, cranial nerves III, IV, VI, mammillary bodies, white matter tracts, and functional cortical areas) to determine the most appropriate surgical approach.</li>
	<li>Select the surgical corridor with the minimal risk of injuries of neural structures39.</li>
	<li>Define the safe resection area for each case, localizing the critical neural structure (such as chiasm, mammillary body) under whose proximity the resection must be arrested to avoid permanent damage39.</li>
	<li>Merge the most relevant MRI sequences and import them into the operative phase&#39;s neuronavigation system.</li>
</ol>
12. Surgery preparation
<ol>
	<li>Induce general anesthesia adopting total intravenous anesthesia with propofol and remifentanil (it has been demonstrated that the other anesthetic agents are among the most critical factors affecting intraoperative monitoring reliability, increasing the false-negative rate), avoiding myorelaxant40.</li>
	<li>Perform oro-tracheal intubation with gauzes in the oropharynx to prevent blood or fluid leakage in the stomach or airways41.</li>
	<li>Set up the neurophysiological monitoring, with continuous recording of motor evoked potentials (MEPs) and somatosensory evoked potentials (SEPs) and free-running electromyography (EMG) for cranial nerves.42</li>
	<li>Import the MRI data, including the tractography reconstructions, in the neuronavigation system (Table of Materials).</li>
	<li>Select the brain surgery electromagnetic registration modality on the neuronavigation system.</li>
	<li>Register the neuronavigation system on the patient, adopting a free-tracking technique or external markers.</li>
	<li>Control the accuracy of the achieved registration, checking the position of external markers (i.e., ear or nose) on the imported MRI; if the result is not acceptable, repeat the registration.</li>
	<li>Place the patient in a semi-sitting position; Mayfield&#39;s use to fix the head is not needed43.</li>
	<li>Administer corticosteroid (endovenous flebocortid, dosage depending on the patient&#39;s weight) and antibiotics (2 g of amoxicillin-clavulanic acid)44.</li>
</ol>
13. Endoscopic endonasal surgery
<ol>
	<li>Start with a 0&#176; endoscope (Table of Materials).</li>
	<li>Harvest the naso-septal flap45.</li>
	<li>Perform an anterior sphenoidotomy, followed by posterior septostomy and ethmoidectomy with preservation of the middle turbinate, when possible43.</li>
	<li>Open the sellar and tuberculum bone41.</li>
	<li>Incise the dura layer with an H-shape, after coagulation of the superior intercavernous sinus41.</li>
	<li>Cleave the tumor by the arachnoidal plane43.</li>
	<li>Centrally debulk the tumor43.</li>
	<li>Remove its capsule from the surrounding diencephalic neural structures, arresting the resection in case of tumor adhesion to eloquent structures visualized under neuronavigation guidance43.</li>
	<li>Explore the surgical cavity with angled optics (Table of Materials)46.</li>
	<li>Ensure hemostasis with bipolar coagulation or hemostatic agents.</li>
	<li>Close the osteo-meningeal opening with an intradural intracranial layer of dural substitute43.</li>
	<li>Place an extradural intracranial layer of dural substitute, scaffolded with abdominal fat and eventually bone (Table of Materials)43.</li>
	<li>Cover the closure with the naso-septal flap43.</li>
</ol>
14. Histological examination
<ol>
	<li>Fix tumor samples with 10% formalin and embed them in paraffin immediately after surgery.</li>
	<li>Cut tissue into sections of 4 &#181;m thickness and stain with hematoxylin and eosin. The histological diagnosis must be based on the most recent version of the WHO classification of brain tumors (2016)47.</li>
	<li>Perform specimen immunohistochemical staining by an automated immunohistochemical staining instrument, using avidin-biotin labeling and diaminobenzidine as a detection reagent. For craniopharyngiomas, adopt anti-beta-catenin, anti-BRAF v600E mutant epitope, and anti-Ki67 antibodies for immunohistochemical staining (Table of Materials).</li>
	<li>Evaluate the Ki-67 index through the manual count of positive tumor cells48.</li>
</ol>
15. Post-surgical patient management
<ol>
	<li>Wake the patient immediately after surgery.</li>
	<li>Restore spontaneous breathing from the mouth by filling nasal cavities with absorbable and non-absorbable material.</li>
	<li>Monitor vital parameters (blood pressure, heart rate, oxygen saturation and consciousness state) for the following 6-12 hours in ICU.</li>
	<li>Restore oral feeding after 12 hours.</li>
	<li>Perform a CT scan after 6-9 hours.</li>
	<li>Maintain bed rest for three days with heparin treatment.</li>
	<li>Control fluid balance every 12 hours and assess serum electrolytes every 24 hours.</li>
	<li>Administer corticosteroid therapy (endovenous flebocortid in the first 24 hours, and then oral cortone acetate 30 +15 mg/day).</li>
	<li>Perform an MRI with/without gadolinium within 72 hours after surgery.</li>
	<li>Discharge the patient on the 4th day.</li>
</ol>
16. Early follow-up
<ol>
	<li>Repeat the complete endocrinological assessment 30 days after surgery43.</li>
	<li>Repeat the ophthalmological assessment three months after surgery43.</li>
	<li>Repeat the neurological physical examination and temperature and sleep-wake rhythms function investigations three months after surgery46.</li>
	<li>Perform the MRI with/without gadolinium three months after surgery46.</li>
</ol>
17. Adjuvant therapy
<ol>
	<li>Evaluate the presence of early tumor progression, and if it is indicated, refer the patient to radiation therapy43.</li>
</ol>
18. Long-term follow-up
<ol>
	<li>Repeat the clinical, endocrinological, and ophthalmological assessments annually43.</li>
	<li>Perform yearly MRI with/without gadolinium: in case of recurrence, the patient can be re-operated on and then referred to radiation therapy or directly referred to radiotherapy43.</li>
</ol>

The authors have nothing to disclose

The application of the presented protocol resulted in a safe and effective treatment of one of the most challenging intracranial tumors such as a craniopharyngioma invading the 3rd ventricle, possibly opening up a new horizon for a lesion that was defined by H. Cushing about a century ago as the most baffling intracranial neoplasm1. The combination of accurate preoperative planning, integrating advanced MRI techniques, and multidisciplinary clinical assessments have permitted us to tail...

The possibility to approach the skull base midline and paramedian regions through an anterior route, adopting the nasal fossae as natural cavities, has a long history, dating back more than one century1. However, in the last 20 years, the visualization and operative technologies have improved enough to expand their possibility of including the treatment of the most complex tumors such as meningiomas, chordomas, chondrosarcomas, and craniopharyngiomas1 due to the (1) introduction of the endoscope, which gives a panoramic and detailed 2D/3D view of these regions to the surgeon, (2) the development of intraoperative neuronavigation systems, and (3) the implementation of dedicated surgical instruments. As painstakingly demonstrated by Kassam et al. and confirmed by multiple reviews and meta-analyses, the advantages of this surgical approach are mainly represented by its chances to resect challenging skull base tumors, avoiding any direct brain retraction or nerve manipulation, thus reducing the risk of surgical complications and long-term neurological and visual sequelae2,3,4,5,6,7,8,9,10,11,12.
For multiple skull base and pituitary-diencephalic tumors, the ideal surgical goal has changed in the last years from the most extensive tumor removal possible to the safest removal with preservation of the neurological functions to preserve the patient&#39;s quality of life3. This limitation could be compensated by innovative and effective adjuvant treatments, such as radiation therapy (adopting massive particles such as proton or carbon ions when appropriate) and, for selected neoplasms, by chemotherapy as inhibitors of the BRAF/MEK pathway for the craniopharyngiomas13,14,15.
However, to pursue these goals, a careful preoperative assessment is crucial, to tailor the surgical strategy to each case&#39;s specific feature2. In most centers, the MRI preoperative protocol is usually performed only with standard structural sequences, which provide the morphological characterization of the lesion. However, with these techniques it is not always possible to assess the anatomical relationship of the tumor with adjacent structures reliably3. Moreover, each patient may present different pathology-induced functional reorganization profiles detectable only with diffusion MRI tractography and functional MRI (fMRI), which can be used to provide guidance both in the surgery planning and in the intraoperative steps16,17.
Currently, fMRI is the most commonly used neuroimaging modality for mapping brain functional activity and connectivity, as guidance for surgical planning18,19 and to improve the patients&#39; outcome20. Task-based fMRI is the modality of choice to identify &#34;eloquent&#34; brain regions that are functionally involved in specific task performance (e.g., finger tapping, phonemic fluency), but is not applicable for the study of skull base tumors.
Diffusion MRI tractography permits in vivo and noninvasive reconstruction of white matter brain connections as well as cranial nerves, investigating the brain hodological structure21. Different tractography algorithms have been developed to reconstruct axonal pathways by linking water molecule diffusivity profiles, evaluated within each brain voxel. Deterministic tractography follows the dominant diffusivity direction, whereas probabilistic tractography evaluates possible pathways&#39; connectivity distribution. Additionally, different models can be applied to evaluate diffusivity within each voxel, and it is possible to define two main categories: single fiber models, such as the diffusion tensor model, where a single fiber orientation is evaluated, and multiple-fiber models, such as spherical deconvolution, where several crossing-fiber orientations are reconstructed22,23. Despite the methodological debate about diffusion MRI tractography, its utility in the neurosurgical workflow is currently established. It is possible to evaluate white matter tract dislocation and distance to the tumor, preserving specific white matter connections. Moreover, diffusion tensor imaging (DTI) maps, especially fractional anisotropy (FA) and mean diffusivity (MD), can be applied to assess microstructural white matter alterations related to possible tumor infiltration and for longitudinal tract monitoring. All these features make diffusion MRI tractography a powerful tool both for pre-surgical planning and intra-operative decision making through neuronavigation systems24.
However, the application of tractography techniques to skull base surgery has been limited by the need for specialized technical knowledge and the time-consuming work-up to optimize diffusion MRI sequence acquisition, analysis protocol, and incorporating tractography results in neuronavigation systems25.Finally, further limitations are due to the technical difficulties extending these analyses from intraparenchymal to extra-parenchymal white matter structures, as cranial nerves. Indeed, only recent studies presented preliminary results attempting to integrate advanced MRI and skull base surgery26,27,28.
The present paper presents a protocol for the multidisciplinary management of pituitary-diencephalic and skull base tumors using diffusion MRI tractography. The implementation of this protocol in the institution resulted from the collaboration between neurosurgeons, neuro-endocrinologists and the neuroimaging team (including clinical and bioinformatics expertise) to offer an effective integrated multi-axial approach to these patients.
In the center, we have integrated multidisciplinary protocols for managing patients with skull base tumors, to provide the most informative description possible, and to tailor and personalize the surgical plan. We show that this protocol can be adopted both in the clinical and the research setting for any patient with a skull base tumor to guide the treatment strategy and to improve the knowledge on the brain modifications induced by these lesions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BRAF V600E-specific clone VE1</td>
			<td>Ventana</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dural Substitute</td>
			<td>Biodesign, Cook Medical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Endoscope</td>
			<td>Karl Storz, 4mm in diameter, 18 cm in length, Hopkins II &#8211; Karl Storz Endoscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immunohistochemical staining instrument&#160;</td>
			<td>Ventana Benchmark, Ventana Medical Systems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MRI</td>
			<td>3T Magnetom Skyra, Siemens Health Care</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neuronavigator</td>
			<td>Stealth Station S8 Surgical Navigation System, MEDTRONIC</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

role of diffusion mri tractography in endoscopic endonasal skull base surgery

Endoscopic endonasal surgery has gained a prominent role in the management of complex skull base tumors. It allows the resection of a large group of benign and malignant lesions through a natural anatomical extra-cranial pathway, represented by the nasal cavities, avoiding brain retraction and neurovascular manipulation. This is reflected by the patients' prompt clinical recovery and the low risk of permanent neurological sequelae, representing the main caveat of conventional skull base surgery. This surgery must be tailored to each specific case, considering its features and relationship with surrounding neural structures, mostly based on preoperative neuroimaging. Advanced MRI techniques, such as tractography, have been rarely adopted in skull base surgery due to technical issues: lengthy and complicated processes to generate reliable reconstructions for inclusion in the neuronavigation system. 
This paper aims to present the protocol implemented in the institution and highlights the synergistic collaboration and teamwork between neurosurgeons and the neuroimaging team (neurologists, neuroradiologists, neuropsychologists, physicists, and bioengineers) with the final goal of selecting the optimal treatment for each patient, improving the surgical results and pursuing the advancement of personalized medicine in this field.

A 55-year-old woman presented with progressive visual deficits. Her medical history was unremarkable. On ophthalmological evaluation, bilateral reduction of visual acuity (6/10 in the right eye and 8/10 in the left eye) was revealed, and the computerized visual field showed complete bitemporal hemianopia. No further deficits were evident on neurological examination, but the patient reported persistent asthenia and an increase in hunger and thirst sensation in the previous 2-3 months, with...

Watch this Scientific Journal Video about Role of Diffusion MRI Tractography in Endoscopic Endonasal Skull Base Surgery at JoVE.com

Role of Diffusion MRI Tractography in Endoscopic Endonasal Skull Base Surgery

We present a protocol to integrate diffusion MRI tractography in patient work-up to endoscopic endonasal surgery for a skull base tumor. The methods for adopting these neuroimaging studies in the pre- and intra-operative phases are described.

Endoscopic endonasal surgery has gained a prominent role in the management of complex skull base tumors. It allows the resection of a large group of ...

role-diffusion-mri-tractography-endoscopic-endonasal-skull-base

Research

JoVE Journal

Neuroscience

3.5K Views.  Center for the Diagnosis and Treatment of Hypothalamic-Pituitary Diseases - Pituitary Unit. We present a protocol to integrate diffusion MRI tractography in patient work-up to endoscopic endonasal surgery for a skull base tumor. The methods for adopting these neuroimaging studies in the pre- and intra-operative phases are described.

Video: Role of Diffusion MRI Tractography in Endoscopic Endonasal Skull Base Surgery

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Optogenetic Functional MRI

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain's global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

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 ...

Endoscopic Endonasal Trans-sphenoidal Approach: Minimally Invasive Surgery for Pituitary Adenomas

Endoscopic endonasal trans-sphenoidal surgery has become the gold standard for the surgical treatment of pituitary adenomas and many other pituitary lesions. Refinements in surgical techniques, technological advancements, and incorporation of neuronavigation have rendered this surgery minimally invasive. The complication rates of this surgery are very low while excellent results are consistently obtained through this approach.
This paper focuses on the step-by-step surgical approach to pituitary adenomas, which is based on personal experience, and details the results obtained with this minimally invasive surgery.

The aim of this paper is to describe the different steps of the endoscopic endonasal approach to the sella turcica.

Endoscopic endonasal trans-sphenoidal surgery has become the gold standard for the surgical treatment of pituitary adenomas and many other pituitary ...

Diffusion Tensor Magnetic Resonance Imaging in Chronic Spinal Cord Compression

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic resonance imaging (MRI) plays an important role in both confirming the diagnosis and evaluating the degree of compression. However, the anatomical detail provided by conventional MRI is not sufficient to accurately estimate neuronal damage and/or assess the possibility of neuronal recovery in chronic spinal cord compression patients. In contrast, diffusion tensor imaging (DTI) can provide quantitative results according to the detection of water molecule diffusion in tissues. In the present study, we develop a methodological framework to illustrate the application of DTI in chronic spinal cord compression disease. DTI fractional anisotropy (FA), apparent diffusion coefficients (ADCs), and eigenvector values are useful for visualizing microstructural pathological changes in the spinal cord. Decreased FA and increases in ADCs and eigenvector values were observed in chronic spinal cord compression patients compared to healthy controls. DTI could help surgeons understand spinal cord injury severity and provide important information regarding prognosis and neural functional recovery. In conclusion, this protocol provides a sensitive, detailed, and noninvasive tool to evaluate spinal cord compression.

Here, we present a protocol for the application of diffusion tensor imaging parameters to evaluate spinal cord compression.

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic ...

Functional MRI in Conjunction with a Novel MRI-compatible Hand-induced Robotic Device to Evaluate Rehabilitation of Individuals Recovering from Hand Grip Deficits

Functional magnetic resonance imaging (fMRI) is a non-invasive magnetic resonance imaging technique that images brain activation in vivo, using endogenous deoxyhemoglobin as an endogenous contrast agent to detect changes in blood-level-dependent oxygenation (BOLD effect). We combined fMRI with a novel robotic device (MR-compatible hand-induced robotic device [MR_CHIROD]) so that a person in the scanner can execute a controlled motor task, hand-squeezing, which is a very important hand movement to study in neurological motor disease. We employed parallel imaging (generalized auto-calibrating partially parallel acquisitions [GRAPPA]), which allowed higher spatial resolution resulting in increased sensitivity to BOLD. The combination of fMRI with the hand-induced robotic device allowed precise control and monitoring of the task that was executed while a participant was in the scanner; this may prove to be of utility in rehabilitation of hand motor function in patients recovering from neurological deficits (e.g., stroke). Here we outline the protocol for using the current prototype of the MR_CHIROD during an fMRI scan.

We performed functional MRI using a novel MRI-compatible hand-induced robotic device to evaluate its utility for monitoring hand motor function in individuals recovering from neurological deficits.

Functional magnetic resonance imaging (fMRI) is a non-invasive magnetic resonance imaging technique that images brain activation in vivo, using endogenous ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed.
Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...