Fiber Connections of the Supplementary Motor Area Revisited: Methodology of Fiber Dissection, DTI, and Three Dimensional Documentation

Podsumowanie

The purpose of this study is to show each step of the fiber dissection technique on human cadaveric brains, the 3D documentation of these dissections, and the diffusion tensor imaging of the anatomically dissected fiber pathways.

Streszczenie

The purpose of this study is to show the methodology for the examination of the white matter connections of the supplementary motor area (SMA) complex (pre-SMA and SMA proper) using a combination of fiber dissection techniques on cadaveric specimens and magnetic resonance (MR) tractography. The protocol will also describe the procedure for a white matter dissection of a human brain, diffusion tensor tractography imaging, and three-dimensional documentation. The fiber dissections on human brains and the 3D documentation were performed at the University of Minnesota, Microsurgery and Neuroanatomy Laboratory, Department of Neurosurgery. Five postmortem human brain specimens and two whole heads were prepared in accordance with Klingler's method. Brain hemispheres were dissected step by step from lateral to medial and medial to lateral under an operating microscope, and 3D images were captured at every stage. All dissection results were supported by diffusion tensor imaging. Investigations on the connections in line with Meynert's fiber tract classification, including association fibers (short, superior longitudinal fasciculus I and frontal aslant tracts), projection fibers (corticospinal, claustrocortical, cingulum, and frontostriatal tracts), and commissural fibers (callosal fibers) were also conducted.

Wprowadzenie

Among the 14 frontal areas delineated by Brodmann, the premotor and prefrontal area that lies in front of the precentral motor cortex has long been considered as a silent module, despite the fact that the frontal lobe plays an important role in cognition, behavior, learning, and speech processing. In addition to the supplementary motor area (SMA) complex, consisting of the pre-SMA and the SMA proper (Brodmann Area; BA 6) that extends medially, the pre-motor/frontal module includes the dorsolateral prefrontal (BA 46, 8, and 9), frontopolar (BA 10), and ventrolateral prefrontal (BA 47) cortices, as well as part of the orbitofrontal cortex (BA 11) on the lateral surface of the brain^1,2.

The SMA complex is a significant anatomical area that is defined by its functions and its connections. The resection and damage of this region causes significant clinical deficits known as the SMA syndrome. The SMA syndrome is an important clinical condition that is particularly observed in frontal glioma cases that contain the SMA complex³. The SMA complex has connections with the limbic system, basal ganglia, cerebellum, thalamus, contralateral SMA, superior parietal lobe, and portions of the frontal lobes via fiber tracts. The clinical effect of damage to these white matter connections may be more severe than to the cortex. This is because the consequences of injury to the cortex can be ameliorated over time due to high cortical plasticity ^{4,5,6,7,8,9,10,11,12,}. Therefore, the SMA regional anatomy and the white matter pathways should be deeply understood, in particular for glioma surgery.

A comprehensive understanding of the anatomy of white matter pathways is important for the wide-spectrum treatment of neurosurgical lesions. Recent studies of the three-dimensional documentation of the anatomical results that were obtained in microsurgery were used to gain a better understanding of the topographical anatomy and the interrelationship of brain white matter pathways^13,14. Therefore, the purpose of this study was to examine the white matter connections of the SMA complex (pre-SMA and SMA proper) using a combination of fiber dissection techniques on cadaveric specimens and magnetic resonance imaging (MRI) tractography and to explain all the methods and principles of both techniques and their detailed documentation.

Planning and Strategy of Study

Prior to performing the experiments, a literature search on the basic principles of fiber dissections, the procedures that need to be applied to specimens before and during dissections, and all connections between SMA regions that have been revealed with dissection and DTI was conducted. The previous studies on the anatomical localization and separation of pre-SMA and SMA-proper regions and on the topographic anatomy of their connections were reviewed.

Protokół

The deceased are included here as a population, although deceased persons are not technically human subjects; human subjects are defined by 45 CF 46 as "living human beings^15,16."

1. Preparation of Specimens

1. Examine 5 formalin-fixed brains (10 hemispheres) and 2 whole human heads.
2. Fix the specimens in a 10% formalin solution for at least 2 months according to Klingler's method¹⁷.
3. Freeze all specimens at -16 ˚C for 2 weeks in accordance with Klinger's method¹⁷.
4. Thaw the specimens under tap water.
5. Perform an extended frontotemporal craniotomy on the cadaveric head to expose the brain.
   1. Place the cadaveric head in a three-pin skull clamp (Material Table).
   2. Make a frontotemporal skin incision with a scalpel.
   3. Remove the skin and muscles using a scalpel, forceps, and scissors.
   4. Make one or more burr holes in the skull until the dura mater is reached; use a drill with a compact speed reducer and a 14 mm cranial perforator attachment at 79,000 rpm (Material Table).
   5. Cut the bone flap and open the skull using a 2 mm x 15.6 mm fluted router with a 2.1 mm pin-shaped burr attachment at a drill speed of 80,000 rpm (Material Table).
6. Remove the dura, arachnoid, and pia mater and dissect using a microdissector under a microscope at 6X to 40X magnification^5,18 (Material Table).

2. Fiber Dissection Technique

NOTE: Perform all dissections under 6X to 40X magnification on a surgical microscope.

1. Perform the fiber dissections in a stepwise manner on each hemisphere, from lateral to medial and medial to lateral.
   1. Decorticate the cerebral cortex using a panfield dissector (Material Table) and remove all frontal cortical tissues to expose the short association fiber tracts, which are U-fibers or intergyral fibers that interconnect neighboring gyri^5,13.
   2. Remove the short association fibers with a panfield dissector and a surgical micro hook by gently trimming under the microscope (Material Table) to reach and expose the long association fibers, which interconnect distant areas in the same hemisphere.
   3. Go deep into the long association fibers to remove the superficial association fibers using a surgical micro hook and a panfield dissector; remove each fiber bundle under a microscope (Material Table) to expose the projection commissural fibers.
   4. View each of the connections of the SMA complex according to the topographical anatomy that was previously defined in the literature^{2,8,18,19,20,21}.
2. Keep all specimens (whole heads and brains) that were used during the dissections in 10% formaldehyde solution (Material Table) between the dissection periods.

3. 3D Photography Technique

1. Use a black color platform during the photography of the specimens.
2. Follow a 3D photography technique²².
   1. Place each specimen in a designed black color platform.
   2. Select a scene with a full-frontal view of the specimen and take one shot by focusing the camera on any point on the specimen close to the center point on the camera screen (instrument table). Use an 18- to 55-mm f/3.5-5.6 SLR lens or a 100 mm f/2.8L macro lens and set the aperture to F29, ISO 100.
   3. Rotate the camera slightly left until the right-most point on the camera screen is the same as the focusing point above. Slide the camera to the right until the middle point on the screen overlaps the original focusing point on the specimen. Focus the camera on this point and take another shot.
   4. Maintain the distance and axis of the camera to the specimen being photographed at constant values.
3. Create a 3D image by using a 3D image generator program (Material Table).
   1. Open the 3D software program.
   2. Choose "Open stereo images from File."
   3. Select the two images (left and right) and make sure the left image is in the left slot and the right image is in the right slot.
   4. Select the "Half color anaglyph RL/2" option and generate the anaglyph in jpeg format.

4. DTI Technique

1. Acquire pre-processed diffusion data utilizing the Human Connectome Project diffusion data²³ by downloading it from the referenced website.
   NOTE: The data is downloaded pre-processed and consisted of the following procedures: The diffusion data was acquired in normal volunteers using a modified 3 T MRI device (instrument table) utilizing a spin-echo echo planar imaging (EPI) sequence with multi-band image acceleration^{24,25,26,27,28}. Relevant sequence parameters include: TR = 5,520 ms; TE = 89.5 ms; FOV = 210 x 180 mm; matrix = 168 x 144; slice thickness = 1.25 mm (voxel size 1.25 x 1.25 x 1.25 mm); multiband factor = 3; and b-values = 1,000 s/mm²(95 directions), 2,000 s/mm²(96 directions), and 3,000 s/mm²(97 directions). The data was then processed utilizing FreeSurfer²⁹and FSL³⁰; the process included eddy current correction, motion correction, b0 intensity normalization, susceptibility distortion correction, and gradient-nonlinearity correction^28,31,32,33. Corresponding T1-weighted MP-RAGE images are also included in the download package. Procedures are documented in the Human Connectome Project procedures manual²³.
2. Post-process the diffusion data using Diffusion Spectrum Imaging (DSI) Studio³⁴to generate an estimated voxel-wise diffusion orientation distribution function (ODF) employing a generalized q-sampling imaging (GQI) algorithm³⁵.
   1. Load the downloaded dataset into the software by selecting "STEP1: Open source images" and selecting the data.nii.gz file.
   2. Select the "STEP2: Reconstruction" button. After verifying the brain mask, proceed to "Step 2" and select "GQI" as the reconstruction method. Select "r^2 weighting" with a "length ratio" of "1.0." Leave the remaining selections as the default.
   3. Select "Run reconstruction."
3. Place appropriate seeds for regions-of-interest to streamline fiber-tracking.
   1. In the "Region Window," click the "Atlas" button to place seeds for the superior longitudinal fasciculus (SLF) I. Select "Brodmann" and add "Region 6" and "Region 7." In the region window, set the "Region 6" type to "seed" and the "Region 7" type to "region-of-inclusions" (ROI).
      1. Select "New Region" in the region window and manually draw an ROI in the most posterior aspect of the superior frontal gyrus in the coronal plane. Perform fiber tracking as described in step 4.4.
   2. Place seeds for SLF II in similar fashion by using "New Region" in the region window and drawing the "seed" region in the posterior aspect of the middle frontal gyrus white matter in the coronal plane. Choose an ROI using "Atlas" (as in step 4.3.1) and Brodmann regions 9, 10, 46, 39, and 19. Perform fiber tracking as described in step 4.4.
   3. Place seeds for SLF III with a "seed" region, using "Atlas" (as in step 4.3.1) in the region window and choosing "Region 40" of the Brodmann atlas and the ROI from "Atlas…" in "Region 40" and "Region 44." Perform fiber tracking as described in step 4.4.
   4. Place seeds for callosal fibers using "New Region" in the region window and drawing a "seed" in the sagittal plane encompassing the corpus callosum. Perform fiber tracking as described in step 4.4.
   5. Place seeds for cingulate fibers using "New Region" in the region window and drawing a "seed" region in the mid-cingulate gyrus on the coronal view. Use "New Region" to draw two ROIs, one in the more anterior cingulate and one in the posterior cingulate gyrus under coronal view. Perform fiber tracking as described in step 4.4.
   6. Place seeds for claustrocortical fibers using "New Region" in the region window and drawing a "seed" in the claustrum with an ROI in the corona radiata using the "Atlas…" function. Select the atlas as "JHU-WhiteMatter-labels-1mm."
      1. Select and add the "Anterior_corona_radiata", "Posterior_corona_radiata," and "Superior_corona_radiata". Draw a region-of-avoidance for all fibers passing through a plane inferior to the level of the claustrum in the axial plane using "New Region" in the region window. Perform fiber tracking as described in step 4.4.
   7. Place seeds for the corticospinal tract using a "seed" from the "Atlas..." function in the region window; select "JHU-WhiteMatter-labels-1mm" and add the "Corticospinal_tract" region. Perform fiber tracking as described in step 4.4.
   8. Place seeds for the frontal aslant tract (FAT) using a "seed" region from the "Atlas..." function in the region window and selecting the Brodmann atlas and "Region 6" ROIs in "Region 44" and "Region 45." Perform fiber tracking as described in step 4.4.
   9. Place seeds for the frontostriatal tract (FST) with a "seed" in "Region 6" using the "Atlas..." function. Insert new regions in the "caudate," "putamen," and "globus pallidus" from the "HarvardOxfordSub" atlas and set the type in the region window to "end."
      ​NOTE: Fiber tracking for FST will be performed by selecting the Region 6 seed and only one of the subcortical seeds per tracking session (i.e., region 6 and the caudate, followed by region 6 and the putamen, and lastly region 6 and the globus pallidus).
      1. Perform fiber tracking as described in step 4.4 for each combination.
4. Perform fiber tracking for each of the above combinations.
   1. In the "Options" window, set the tracking parameters as: termination index of qa of 0.08, angular threshold of 75, step size of 0.675, smoothing of 0.2, minimum length of 20 mm, and maximum length of 200 mm. Select the seed orientation as "All," the seed position as "Subvoxel," and randomize seeding as "On." Use trilinear direction interpolation with a streamline (Euler) tracking algorithm. For each combination of regions above, choose "Run tracking" in the "Fiber Tracts" window.
      NOTE: Due to the randomized nature of the tracking, clear "false fibers" are identified and selectively removed, with regions-of-avoidance drawn by hand as a "New Region."
5. Affine register the brain-extracted T1-weighted 3D MP-RAGE scan provided in the Human Connectome Project data set to the diffusion data using the "Slices -> Insert T1/T2 Images" function of DSI-Studio. Generate a surface rendering of the brain by selecting "Slices -> Add Isosurface." Use a "threshold" of 665.

Wyniki

The SMA complex is situated in the posterior part of the superior frontal gyrus. The borders of the SMA complex are the precentral sulcus posteriorly, the superior frontal sulcus inferior-laterally, and the cingulate sulcus inferior-medially¹⁸. The SMA complex consists of two parts: the pre-SMA anteriorly and the SMA proper posteriorly¹⁸. There are differences in terms of white matter connections and function between these two parts

Dyskusje

The Importance of and Study Techniques for the White Matter Pathways

The cerebral cortex is accepted as a principal neural structure associated with 2.5 million years of human life. Approximately 20 billion neurons have separated into various parts based upon morphological and cellular specification⁴⁰. The architecture of each of these cortical parts has been functionally sub-grouped, such as sensorimotor sense and movement, emotional experience, and complex reasoning. It ...

Materiały

Name	Company	Catalog Number	Comments
%4 Paraformaldehyde Solution	AFFYMETRIX, Inc.	2046C208	used to fixation
Freezer	INSIGNA	NS-CZ70WH6	used to freez
Panfield Dissector	AESCULAP	FD305	used to dissection
Surgical Micro Scissor	W. Lorenz	04-4238	used to miscrodissection
Surgical Micro Hook	V. Mueller	NL3785-009	used to miscrodissection
MICRO VESSEL STRETCHER/DILATOR	W. Lorenz	04-4324	used to miscrodissection
Emax2 SC 2000 Electric Console	Anspach Companies	SC2102	used to craniatomy
Drill Set	Anspach Companies	NS-CZ70WH6	used to craniatomy
20-1000 operating microscope	Moeller-Wedel,Germany	FS 4-20	used to miscrodissection
Canon EOS 550D 18 MP CMOS APS-C Digital SLR Camera	Canon Inc.	DS126271	used to take photos
EF 100mm f/2.8L IS USM Macro Lens	Canon Inc.	4657A006	used to take photos
MR-14EX II Macro Ring Lite (Flash)	Canon Inc.	9389B002	used to take photos
Tripod	Lino Manfrotto	322RC2	used to take photos
MAYFIELD Infinity Skull Clamp	Integra Inc.	A0077	used to fix the head
Modified Skrya 3T "Connectome" Scanner	Siemens Company, Inc.	A911IM-MR-15773-P1-4A00	used to scan DTI
XstereO Player	Yury Golubinsky	Version 3.6(22)	used to create anaglyphs
EF-S 18-55mm f/3.5-5.6 IS II SLR Lens	Canon Inc.	2042B002	used to take photos
Scalpel	6B INVENT	7-104-L	used to make incision
Compact  Speed Reducer	Anspach Companies	CSR60	used to make burr hole
14 mm Cranial Perforator	Anspach Companies	CPERF-14-11-3F	used to make burr hole
2 mm x 15.6 mm Fluted Router	Anspach Companies	A-CRN-M	used to make craniotomy
2.1 mm Pin-shaped Burrs	Anspach Companies	03.000.130S	used to make craniotomy