This work was supported by NIH Grants R21NS076176, R01NS089069, R01EY028535, and NSF Grant BCS-1349042 to BZM, and by a University of Rochester Center for Visual Science predoctoral training fellowship (NIH training Grant 5T32EY007125-24) to FEG. We are grateful to Keith Parkins for his work on the development of StrongView, which was supported by core grant P30EY00131 to the Center for Visual Science at the University of Rochester Medical School. The Program for Translational Brain Mapping at the University of Rochester was established, in part, with support from Norman and Arlene Leenhouts, and with a grant from the Wilmot Cancer Institute to Drs. Kevin Walter and Bradford Mahon. Information about the Program for Translational Brain Mapping at the University of Rochester Medical Center can be found at: www.tbm.urmc.edu.

The activities shown in the video and described herein fall within a greater-than-minimal-risk IRB at the University of Rochester Medical Center.
1. Recruitment
<ol>
	<li>Establish a high through-put program for pre-operative cognitive and MRI-based assessment to catch patients from all referring providers in a timely and efficient manner. Involve the administrative and clinical staff in the broader effort. 
	NOTE: A concrete step that has proved effective was the establishment of a group email list that is automatically sent by the attending surgeon (or someone on their support staff) when a new patient presents to clinic who may be a candidate for recruitment into the Brain Mapping Program.</li>
</ol>
2. Pre-Operative MRI mapping
<ol>
	<li>Acquire MRI data on a 3T MRI scanner with a 64-channel head coil at the Center for Advanced Brain Imaging and Neurophysiology (formally known as the &#39;Rochester Center for Brain Imaging&#39;) at the University of Rochester Medical School. Use standard sequences for BOLD MRI and DTI allowing full brain imaging, as described in prior publications7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25.</li>
	<li>Monitor fixation, and record respiration and heart rate collected during all fMRI for regression of noise confounds26,27. 
	NOTE: Over the past 10 years, we have developed a library of functional MRI experiments to map language (spoken, auditory, single words, whole sentences), motor function (from intransitive finger, tongue and foot movements to high level transitive actions), music ability, mathematics and number knowledge, and basic sensory function (e.g., retinotopic mapping to map of low level visual processing11,14,24). All experiments, materials, and analysis scripts are available at www.openbrainproject.org.</li>
</ol>
3. Neuropsychological testing
<ol>
	<li>Take care during all cognitive testing to ensure that patients are comfortable, ensured using an ergonomically optimized setup (Figure 1) and by building frequent breaks (every 8 min) into the structure of all tests.</li>
	<li>Have all low grade tumor patients complete the following tests 1 month before surgery, 1 month after surgery, and 6 months after surgery (tests 12 and 13 are completed only at the pre-operative and 6 months post-operative time points)28,29,30,31,32.
	<ol>
		<li>Spontaneous Speech (Cookie Theft Picture33, Cinderella Story34,35,36).</li>
		<li>Category Fluency (actions, semantic categories, words starting with F, A, S).</li>
		<li>Word Reading and Repetition (nouns, verbs, adjectives, non-words, matched on length and frequency).</li>
		<li>Snodgrass Object Naming (n = 26037).</li>
		<li>Auditory Naming (n = 6038).</li>
		<li>High-Cloze Sentence Completion (30 min).</li>
		<li>Birmingham Object Recognition Battery (BORB, including Length | Size | Orientation | Gap Matching | Overlapping Figures | Foreshortened Views | Object Reality Decision39).</li>
		<li>Auditory Minimal Pair Discrimination (e.g., pa vs. da, ga vs. ta31,40).</li>
		<li>Sentence Picture Matching (including reversible passives40).</li>
		<li>Color Naming and Farnsworth Munsell Hue Sorting41.</li>
		<li>Cambridge Face Test30,42.</li>
		<li>California Verbal Learning Test (43)</li>
		<li>Weshler IQ (44,45,46). The key measures to evaluate language outcome are tests 4-6; characterizing broader abilities ensures impairments on naming tests are not due to general performance decline47. 
		NOTE: In the past, we have used a combination of software presentation platforms to control stimulus presentation and response recording during pre- and post-operative testing. We are currently designing a single plug-and-play platform to support all cognitive testing (pre, intra- and post-operative testing) as well as stimulus presentation and response recording during functional MRI (see below for description of StrongViewTM). StrongView, together with built-in neuropsychological tests, will be available for download (open license) at www.openbrainproject.org.</li>
	</ol>
	</li>
</ol>
4. Neuroanesthesia and ergonomics of intraoperative language mapping
<ol>
	<li>Use anesthetic techniques for awake craniotomies48,49,50; at the University of Rochester, awake craniotomies are typically performed using an asleep-awake-asleep approach.</li>
	<li>Avoid premedications such as anticonvulsants and anxiolytics as they can impair cognitive function and contribute to emergence delirium.</li>
	<li>Apply standard monitors (EKG, NIBP, pulse oximetry) and induce general anesthesia with intravenous fentanyl (0.5 mg/kg), lidocaine (1-1.5 mg/kg) and propofol (1-2 mg/kg).</li>
	<li>Use a supraglottic airway for mechanical ventilation.</li>
	<li>Position the patient laterally or semi-laterally with the head secured in a pinned frame; as described in the video, patient positioning depends on the location of the lesion and the planned craniotomy window, while also taking into consideration that types of cognitive testing the patient will be asked to perform once awake during the surgery.</li>
	<li>Apply analgesia at the pin and incision site (30 mL of 0.5% Lidocaine, 30 mL of 0.5% Sensorcaine plain, 6 mL of sodium bicarbonate). During this period, position the testing equipment (small monitor, video cameras, directional mics).</li>
	<li>Determine the size of the craniotomy window by multiple factors, which vary in their weighting according to the results of pre-operative clinical mapping of the patient&#39;s brain, functional brain mapping studies, and the plan for intra-operative mapping. In the case described in the video, the attending surgeon (Dr. Pilcher) chose a large craniotomy in order to have full access to map positive language and motor sites in the dominant hemisphere.</li>
	<li>At the beginning of the awake-phase, discontinue sedation (local analgesics are applied prior to incision).</li>
	<li>Remove the supraglottic airway once the patient regains consciousness. There is no or minimal sedation during the awake phase.</li>
	<li>Use electrocorticography (ECoG) to monitor after-discharges (subclinical epileptiform discharges induced by cortical stimulation) to ensure that DES levels are set at just below the after-discharge threshold. The DES mapping procedure initiates by finding the after-discharge threshold, and adjusting stimulation amplitude (in steps of .5 milliamp).</li>
	<li>Adjust stimulation amplitude throughout the mapping session (2 to 15 mA) at the discretion of the attending surgeon. Patients view stimuli on a monitor and can speak and move their forearms and hands.</li>
</ol>
5. Procedures for acquiring research-grade data during intraoperative direct electrical stimulation mapping
<ol>
	<li>Run all intraoperative cognitive testing on a custom-built hardware/software system called &#39;StrongView&#39;,&#160;available at www.openbrainproject.org. The hardware footprint is self-contained on a small cart, and is outfitted with an independent backup battery power source, speakers, keyboard and touch display. The person charged with running the cognitive testing can start, stop and pause stimulus presentation, while continuously recording (audio and video) during the case.</li>
	<li>Use an audio system on the cart such that a directional microphone that is trained on the patient&#39;s mouth, which feeds through a splitter. One channel coming out of the splitter goes through an amplifier and directly to a speaker. This allows surgeons and researchers to easily hear the patient&#39;s responses against the background noise of the operating room with zero perceptible delay (i.e., eliminating &#39;echo&#39; effects). The second channel from the splitter goes to the PC on the mobile cart, where it is time-stamped, recorded and stored (these files are used for offline analysis). StrongView also has a separate (stand-alone) audio system that consists of a second directional microphone also trained on the patient, a directional microphone trained on the surgeons, and a &#39;noise&#39; microphone in a corner of the operating room to sample room tone for subtraction from the main audio files. Those three audio channels feed to a MIDI, and to a second computer which records each channel separately. This second audio system provides redundancy should the primary system fail, all verbal responses of the patient will be available for offline analysis.</li>
	<li>Attach a commercially available ether screen L-bracket to the operating room (OR) table using an OR table clamp. Attach articulating arms (e.g., Manfrotto 244 Variable Friction Magic Arms) to the ether screen L-bracket, and those articulating arms support the patient monitor, directional microphones, video camera trained on the patient&#39;s face, and an auxiliary monitor to allow a research team member or operating room nurse to easily see what the patient sees while interacting with the patient.</li>
	<li>Run all necessary cables for the screens, microphones, and camera along the arm and protect by plastic tubing secured with Velcro. 
	NOTE: None of this equipment needs to be sterilized as it is (only ever) on the non-sterile side of the field (Figure 1). This manner of supporting stimulus presentation and response recording equipment provides maximal flexibility to take into account the differing ergonomics of cognitive testing according to patient positioning which varies case by case, yet provides a reliable and stable platform on which to attach equipment. Also, and importantly, because all monitors, microphones and cameras are attached to the OR table via single device (ether screen L-bracket), if the positioning of the table is adjusted during the case this does not affect the testing setup. (Note that the setup shown in Figure 1 is from an earlier generation setup in which a floor-mounted stand supported the patient screen, microphone and video camera; that floor mounted stand has been replaced since 2018 with the ether screen L-bracket). Also, and importantly for patient safety, the entire setup for cognitive testing can be broken down in less than 20 seconds during the case should an emergent situation present itself that mandates full and unobstructed access to the patient (e.g., to the patient&#39;s airway).</li>
	<li>The heart of StrongView is a flexible software system for i) presenting stimuli (visual, auditory) to patients and recording patient responses (verbal, button response, video), ii) temporally registering all experimentally-relevant events and measures (stimulus on, ECoG, contact with brain of direct electrical stimulator probe, patient responses); iii) and communication with cranial navigation systems to obtain the 3 dimensional coordinate for each application of direct electrical stimulation. StrongView allows on-line re-calibration of experimental variables such as stimulus duration, inter-stimulus-intervals, randomization, number of repetitions or blocks of stimuli, and control of the patient video and audio channels. StrongView streams the patient video camera, the online ECoG data, and the stimulus that the patient is currently seeing/hearing to a desktop display, which is also mirrored on a large monitor that is in the line of sight of the surgeon.</li>
	<li>Attach a photodiode to the patient monitor and feed into an open channel on the ECoG amplifier. This provides a temporal sync between presentation of each stimulus and ECoG for offline analysis.</li>
	<li>Use cranial navigation hardware and software (at the University of Rochester, BrainLab Inc., Munich, Germany) in all cases by the surgical team for intra-operative cranial navigation based on pre-operative MRI. This is an optical system consisting of a set of cameras that view the operating field and register the patient&#39;s head via a fixed registration star that is affixed to the operating table (see Figure 1).
	<ol>
		<li>Specifically, after the patient is set in the headholder, but before draping, use the facial physiognomy of the patient to register the patient&#39;s head to the preoperative MRI. This allows pre-operative MRI (functional and structural) to be brought into direct alignment with the patient&#39;s brain on the operating table.</li>
		<li>Attach a second (much smaller) registration star to the bipolar stimulator (see Figure 1) and use to register the stimulator&#39;s length and position in the field. This enables the research team to acquire the precise location of each point of stimulation as well as the margins of the resection, relative to pre-operative MRI. As noted above, StrongView is connected with the cranial navigation system (at University of Rochester, BrainLab, connection via IGT link) to allow for real time streaming (and time stamping) of the coordinates of direct electrical stimulation mapping. StrongView is currently being developed to interface with other cranial navigation systems (e.g., Stryker). 
		NOTE: Aspects of StrongView that support administration and data collection during cognitive and fMRI experiments, together with a library of tests, will be available (open access) at OpenBrainProject.org. Beta versions are available prior to the full release by contacting the corresponding author. The entire StrongView suite, which includes hardware systems to integrate with Electrocorticography and cranial navigation software, is available to clinicians and scientists by contacting the corresponding author. These data acquisition tools will be confluent with a post-processing pipeline and open data consortium, to be launched in 2020 at OpenBrainProject.org.</li>
	</ol>
	</li>
</ol>

A provisional patent (U.S. Provisional Patent Number 62/917,258) was filed&#160;11/30/18 for &#34;StongView: An integrated hardware/software system to facilitate cognitive testing during awake brain surgery and to support real-time analyses in the service of predicting patient outcome.&#34;

The knowledge gained from the experience of establishing the Program for Translational Brain Mapping at the University of Rochester can be distilled down into two core elements. First,&#160;structured channels of communication were established among cognitive scientists, neuro-oncologists, neuropsychologists, epileptologists, neurophysiologists, neuro-anesthesiologists, neurosurgeons and their respective supporting technicians and administrative support. This allows patients, including urgent high-grade tumor patients, to be referred for pre-operative evaluation with sufficient time to turn analyses around to surgeons prior to the procedure. The second component critical to the success of the Brain Mapping Program has been to fold in training opportunities for undergraduate students, graduate (MS, PhD) students, medical students, as well as neurosurgery, neurology and neuroradiology residents and fellows. The combination of those two elements serve to engage all clinical providers with the scientific objectives of the Brain Mapping Program, and ensures that basic science objectives are intertwined with the clinical goal of optimizing the outcome of every patient.

Neurosurgical interventions to remove brain tumors or epileptogenic tissue adjacent to brain areas that support critical cognitive functions must balance the clinical objective of the surgery (remove as much tumor, or epileptogenic tissue as possible) against damage to healthy tissue that could cause neurologic deficits. In the context of brain tumor surgery, this balance is referred to as the onco-functional balance. On the &#39;onco&#39; side of the balance, surgeons want to remove as much of the tumor as possible, as rates of &#39;gross total tumor resection&#39; are linked to longer survival1,2. On the &#39;functional&#39; side, removal of tumors can damage cortical and subcortical substrates of cognition; post-operative difficulties can involve language, action, vision, hearing, touch or movement, depending on the neural system(s) affected. The onco-functional balance is critically important because increased morbidity is associated with i) lower quality of life, ii) increased post-operative complications that can increase mortality (e.g., patients who can no longer move are at a higher risk of blood clots3,4). The tension inherent in the &#39;onco-functional&#39; balance in the setting of brain tumor surgery translates as well to epilepsy surgery &#8212; there the balance is between the clinical objective of removing all tissue that is generating seizures, while not removing tissue that supports critical functions.
At a broad level, functional neuroanatomy is highly stereotyped from individual to individual. However, there can be a high degree of individual variability in the precise (i.e., mm to mm) location of higher cortical functions. In addition, it is generally recognized that the presence of cortical or subcortical pathology can spur cortical reorganization, although the principles that drive such reorganization are poorly understood5. Neurosurgical interventions proceed millimeter by millimeter. It is thus critical to map each patient&#39;s brain, in detail and with sensitivity and precision, in order to understand which regions in that specific patient support which sensory, cognitive and motor functions6.
The Program for Translational Brain Mapping at the University of Rochester has been engineered to meet the needs of personalized brain mapping in the setting of a high through-put practice spanning multiple academic surgeons. The synergistic goals of the Brain Mapping Program are to i) use the tools of cognitive neuroscience to advance personalized neuromedicine, in the form of patient-specific functional brain maps, and ii) use the clinical preparation of neurosurgical interventions to test mechanistic hypotheses about how the human brain functions.

translational brain mapping at the university of rochester medical center: preserving the mind through personalized brain mapping

The Translational Brain Mapping Program at the University of Rochester is an interdisciplinary effort that integrates cognitive science, neurophysiology, neuroanesthesia, and neurosurgery. Patients who have tumors or epileptogenic tissue in eloquent brain areas are studied preoperatively with functional and structural MRI, and intraoperatively with direct electrical stimulation mapping. Post-operative neural and cognitive outcome measures fuel basic science studies about the factors that mediate good versus poor outcome after surgery, and how brain mapping can be further optimized to ensure the best outcome for future patients. In this article, we describe the interdisciplinary workflow that allows our team to meet the synergistic goals of optimizing patient outcome and advancing scientific understanding of the human brain.

Figure 2,&#160;Figure 3, and&#160;Figure 4&#160;present representative results of pre-operative functional and structural mapping for three patients with tumors that were adjacent to eloquent regions of the brain. The findings shown in Figure 2,&#160;Figure 3, and&#160;Figure 4&#160;are intended to be illustrative (rather than an exhaustive summary) of the types of maps that are generated for each patient. Details on the cases presented in Figure 2,&#160;Figure 3, and&#160;Figure 4&#160;can be found in: Figure 2 (Chernoff, Teghipco, Garcea, Sims, Belkhir,&#160; Paul, Tivarus, Smith, Hintz, Pilcher, Mahon, in press51),&#160;Figure 3 (Chernoff, Sims, Smith, Pilcher and Mahon, 201952), and Figure 4 (Garcea et al., 201716). An important consequence of consecutive recruitment of glioma patients into a uniform protocol is that it makes possible group-level analyses that evaluate the effect of brain tumors on network function and organization. As an example of this type of analyses, Figure 5 presents results from a recent study 14&#160;that found that tumors in left parietal cortex modulated neural responses to &#39;tools&#39; (small manipulable objects) in the temporal lobe, an instance of a more general phenomenon referred to as dynamic diaschesis53.&#160;
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59592/59592fig1.jpg" /> 
Figure 1.&#160;Overview of Equipment used for extra-operative and intra-operative cognitive testing. (A) Example setup for high through-put cognitive neuropsychological testing as implemented by the Program for Translational Brain Mapping in the Department of Neurosurgery at the&#160;University of Rochester Medical Center. Key elements for ensuring that all recruited patients are able to complete all planned tests include: i) a place for patients to sit and complete testing that is fully adjustable to each patient&#39;s size, including a chair specifically designed to reduce fatigue, and ii) locating cognitive/behavioral testing physically adjacent to the MRI. These elements allow patients to visit the facility and complete their functional and structural MRI within the same session as core behavioral data are measured. Participants complete more trials with better performance if they are comfortable, especially for older participant populations with other comorbidities that can make sitting for prolonged periods uncomfortable. (B) Equipment used during intraoperative mapping. The image at the left shows a patient before being draped (right is after draping). Before draping, the cognitive science team sets up their equipment, including audio and video recorders of the patient, a monitor positioned in front of the patient&#39;s line of sight, and a second monitor positioned so that the person working with the patient can easily see the stimulus at which the patient is currently looking (see &#39;Procedure&#39; for details). (C) Bipolar Stimulator with registration star attached to record locations of intra-operative stimulation in preoperative MRI DICOM space. Usually at the point in the surgery at which the dura has been retracted and the patient is being awoken from general anesthesia, there are a few minutes in which to register the bipolar stimulator to the field. This must be done by a team member who is scrubbed in to the case (i.e., either attending or resident surgeon or a scrub tech/nurse). It is accomplished by attaching a small registration star to the bipolar stimulator and following the instructions in the cranial navigation system to register a new instrument on the field. <a href="https://www.jove.com/files/ftp_upload/59592/59592fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59592/59592fig2.jpg" /> 
Figure 2. Pre-operative functional MRI and Diffusion Tensor Imaging (DTI) in patient AH&#160;with a left inferior parietal glioma that infiltrated the arcuate fasciculus. (A) Pre-operative T1 MRI and 3D reconstruction of the left arcuate fasciculus and glioma. The arcuate fasciculus is shown in orange at a 5% threshold with the tumor reconstructed in blue. (B) Pre-Operative functional MRI. The patient completed several sessions of functional MRI that were each designed to map a function that was anticipated to be adjacent to the area of surgical intervention. All maps are thresholded at FDR q &#60; .05 or better. In blue are voxels that exhibit differential neural responses when naming tools compared to animals; in line with prior studies from our lab using the same stimuli, a robust network is identified involving premotor, parietal, and lateral and ventral temporal areas7,8,9,10,14,15,17,18,19,20,21,22,28. The patient was also asked to carry out a numerosity task in which he had to judge which of two clouds of dots had more dots; the two clouds of dots could either have a similar number of dots (hard comparison, ratio = 0.8) or very different numbers of dots (easy comparison, ratio = 0.25). In green are voxels that exhibit differential neural responses when carrying out the task over hard ratio stimuli (ratio = .8) compared to easy stimuli (ratio = .25 54,55). The patient was also asked to move his hands and feet (either flex/extension or rotate25). In red are voxels that exhibited differential neural responses to movements of the right hand compared to movements of the right foot. Finally, the patient was asked to generate as many items as he could think of in 30 seconds from various categories (e.g., &#39;things you do in the kitchen&#39;, &#39;animals&#39;, words that start with &#39;F&#39;, etc.). In purple are voxels that exhibited differential neural activity for overt word production compared to fixation/rest. <a href="https://www.jove.com/files/ftp_upload/59592/59592fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59592/59592fig3.jpg" /> 
Figure 3.&#160;Pre-Operative white matter tractography of the Frontal Aslant Tract and adjacent u-shaped fibers. Prior experience in the Program for Translational Brain Mapping&#160;(Chernoff et al., 201756) with brain mapping in patients with gliomas adjacent to the frontal aslant tract demonstrated that (even partial) transection of this pathway can be associated with dysfluencies in spontaneous speech, while repetition of spoken language can remain intact. That prior experience was used to inform pre-operative mapping of the frontal aslant tract in patient AI11. (A) Coronal slices showing the frontal aslant tract (blue-light blue) and u-shaped fibers (red-yellow). The frontal aslant tract passes just anterior and medial to the glioma. (B) 3D Rendering of frontal aslant tract (blue) and tumor (red) from multiple perspectives. The pre-operative anatomical studies (Panels A and B) indicated that at the end of the tumor resection, it would be possible to define the anterior margin of tumor using direct electrical stimulation mapping. We thus designed a new language task based on our prior experience, specifically to test whether stimulation of the frontal aslant tract disrupted sentence production at the boundaries of grammatical phrases. (C) Direct electrical stimulation of the frontal aslant tract disrupts sentence production differentially at the boundaries of grammatical phrases. The screenshot (Panel C, left) from the video shows the patient, the stimulus with which he was presented, the surgeon&#39;s hand holding the bipolar stimulator in contact with the frontal aslant tract at the anterior margin of the tumor, and the location in coronal and sagittal slices of the current stimulation location (red dot) in relation to the frontal aslant tract (blue). The patient&#39;s task was to describe the spatial relation of the target shape in relation to the location of a reference shape (for the trial shown, the correct response would be: &#34;The red square is below the red diamond&#39;). We found that stimulation of the frontal aslant tract disrupted sentence production, and differentially so at the start of new grammatical phrases (Panel C, graph at right; for video of the intraoperative mapping procedure in this patient, see www.openbrainproject.org). This observation motivates a novel hypothesis about the role of the frontal aslant tract in sentence production: the Syntagmatic Constraints on Positional Elements (SCOPE) hypothesis11. <a href="https://www.jove.com/files/ftp_upload/59592/59592fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59592/59592fig4.jpg" /> 
Figure 4. Pre-Operative functional and structural MRI and intraoperative direct electrical stimulation mapping in a professional musician with a glioma in the right posterior temporal lobe. (A) Pre-operative fMRI mapping of high-level visual processing, language production, and tool knowledge. The tumor, shaded yellow, was in the right temporal lobe, visible through the right superior temporal sulcus (sulci slightly expanded to facilitate visualization). Because the tumor was located close to motion processing areas in lateral temporal cortex, we localized MT/V5 by comparing neural activity when the patient attended to arrays of moving dots to neural activity elicited by stationary dots; voxels exhibiting differential neural responses for motion compared to static dots are plotted on the purple-white color scale (we are grateful to Duje Tadin for assistance with developing this functional localizer). As for all other cases studied in the Program for Translational Brain Mapping (e.g., Figure 2, Figure 3), voxels exhibiting differential neural responses for naming common pictures are compared to a baseline of viewing phase-scrambled versions of the same images; this is plotted on the green-white color scale. That contrast identified bilateral lateral occipital complex, bilateral middle/superior temporal gyrus, and motor cortex (associated with speech motor activity). Also as in Figure 2, voxels exhibiting differential neural responses when naming &#39;tools&#39; were found in the left inferior parietal lobule, bilateral superior parietal/dorsal occipital cortex, and the left posterior middle/inferior temporal gyrus (blue-white color scale). Finally, and again as in Figure 2, the patient was asked to complete a verbal fluency word production task. Voxels associated with word generation compared to a resting baseline are plotted on the red-white color scale and were found in the left inferior frontal gyrus (Broca&#39;s area), superior temporal/inferior parietal cortex, and the speech motor system. (B) The patient completed multiple functional MRI experiments pre-operatively specifically to map music processing. In one experiment, modeled after prior work from Greg Hickok&#39;s lab57, the patient heard short piano melodies and had to hum the melody back, or heard short sentences and had to repeat the sentences back. Plotted on the brain on the red-purple color scale are voxels that exhibited differential neural activity for music than for language. Four Eastman School of Music Graduate students completed the same fMRI experiment; the border of the region identified for the same functional contrast in the matched healthy controls is plotted in green outline. In addition, 10 other neurosurgery patients completed the same experiment, also in the preoperative phase of their treatment. While the proximate goal in those 10 patients was to identify language-responsive areas (thorugh the contrast of language &#62; music), the contrast of music&#62;language identifies a very similar region of the right superior temporal gyrus (borders of the functional region from the 10 control neurosurgery patients are drawn in light blue). (C) Pre-operative probabilistic tractography over DTI data showing the right acoustic radiations and arcuate fasciculus in relation to patient AE&#39;s tumor (5% threshold, overlaid on native T2-weighted image). (D) During his surgery, patient AE performed the same task as during fMRI in which he had to listen to short piano melodies and hum them back, or a short sentence and repeat it back. It was found that direct electrical stimulation to the right posterior superior temporal gyrus disrupted performance in the repetition task when performed over melodies (for some trials), but did not affect performance (on any trials) for the same repetition task performed over sentences (see www.openbrainproject.org for videos of intraoperative music mapping). <a href="https://www.jove.com/files/ftp_upload/59592/59592fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59592/59592fig5.jpg" /> 
Figure 5.&#160;Demonstration of domain-specific diaschesis: Analysis of the relation of lesion location and stimulus-elicited neural activity across a group of glioma patients studied pre-operatively in the Program for Translational Brain. An important consequence of administering a common set of functional MRI and behavioral studies to all patients who go through the Program for Translational Brain Mapping at the University of Rochester Medical Center is the opportunity to carry out group-level analyses on larger sets of consecutively studied patients. As an example, Figure 5 shows the results of a test of the basic science hypothesis that neural responses to &#39;tools&#39; in the temporal lobe are modulated online by inputs from parietal cortex. If that hypothesis is correct, then lesions (tumors) in parietal cortex should alter neural responses in the temporal lobe to &#39;tools&#39;, and variance across patients in neural activity to &#39;tools&#39; in the temporal lobe should be correlated with the presence of lesions (tumors) in parietal cortex. (A) Lesions to parietal cortex are predicted at the group level (logistic regression) from variance across patients in neural responses in the medial fusiform gyrus on the ventral surface of the temporal lobe. (B) Neural responses to tools in the medial fusiform gyrus are predicted at the group level (logistic regression) from variance in whether lesion/tumor involves the anterior Intraparietal Sulcus (aIPS). The findings summarized in panels A and B represent an instance of dynamic diaschesis53, in this case &#39;domain-specific&#39; dynamic diaschesis, because the relation of lesion location to neural activity is modulated by the type of stimulus being processed (i.e., the relation is present for tools, and not for places, face or animals)-for full details see Garcea and colleagues14. <a href="https://www.jove.com/files/ftp_upload/59592/59592fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping

This article provides an overview of a multi-modal brain mapping program designed to identify regions of the brain that support critical cognitive functions in individual neurosurgery patients.

The Translational Brain Mapping Program at the University of Rochester is an interdisciplinary effort that integrates cognitive science, neurophysiology, ...

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45.0K Views.  University of Rochester Medical Center. Het doel van alle hersentumor chirurgie is echt om wat we noemen een maximale veilige chirurgische resectie te krijgen. Dat betekent dat we alle tumoren willen verwijderen, maar we willen geen negatieve effecten hebben op het omringende normale hersenweefsel. Voorafgaand aan een hersentumor operatie in welsprekende cortex, de patiënt wordt toegelaten tot het programma voor translationele hersenen mapping.Alle informatie die we krijgen van de functionele MRI-scan wordt daadwerkelijk ge...

Video: Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Medicine

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Isolation and Characterization of Bone Marrow Microenvironment in Myeloid Neoplasms

Identifying Bone Marrow Microenvironmental Populations in Myelodysplastic Syndrome and Acute Myeloid Leukemia

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and fibroblasts, which provide support for hematopoietic stem cells (HSCs). In addition to supporting normal HSCs, the bone marrow microenvironment also plays a role in the development of hematopoietic stem cell disorders, such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). MDS-associated mutations in HSCs lead to a block in differentiation and progressive bone marrow failure, especially in the elderly. MDS can often progress to therapy-resistant AML, a disease characterized by a rapid accumulation of immature myeloid blasts. The bone marrow microenvironment is known to be altered in patients with these myeloid neoplasms. Here, a comprehensive protocol to isolate and phenotypically characterize bone marrow microenvironmental cells from murine models of myelodysplastic syndrome and acute myeloid leukemia is described. Isolating and characterizing changes in the bone marrow niche populations can help determine their role in disease initiation and progression and may lead to the development of novel therapeutics targeting cancer-promoting alterations in the bone marrow stromal populations.

Author Spotlight: Analyzing Bone Marrow Microenvironment in Murine Hematological Malignancies 

Here a detailed protocol to isolate and characterize bone marrow microenvironmental populations from murine models of myelodysplastic syndromes and acute myeloid leukemia is presented. This technique identifies changes in the non-hematopoietic bone marrow niche, including the endothelial and mesenchymal stromal cells, with disease progression.

The bone marrow microenvironment consists of distinct cell populations, such as mesenchymal stromal cells, endothelial cells, osteolineage cells, and ...

Cancer Research

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Murine Flexor Tendon Injury and Repair Surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

Cantilever Bending of Murine Femoral Necks

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and therapies, with biomechanical testing as a primary outcome measure. However, traditional biomechanical testing focuses on torsion or bending tests applied to the midshaft of the long bones. This is not typically the site of high-risk fractures in osteoporotic individuals. Therefore, a biomechanical testing protocol was developed that tests the femoral necks of murine femurs in cantilever bending loading to replicate better the types of fractures experienced by osteoporosis patients. Since the biomechanical outcomes are highly dependent on the flexural loading direction relative to the femoral neck, 3D printed guides were created to maintain a femoral shaft at an angle of 20&#176; relative to the loading direction. The new protocol streamlined the testing by reducing variability in alignment (21.6&#176; &plusmn; 1.5&#176;, COV = 7.1%, n = 20) and improved reproducibility in the measured biomechanical outcomes (average COV = 26.7%). The new approach using the 3D printed guides for reliable specimen alignment improves rigor and reproducibility by reducing the measurement errors due to specimen misalignment, which should minimize sample sizes in mouse studies of osteoporosis.

The present protocol describes the development of a reproducible testing platform for murine femoral necks in a cantilever bending set-up. Custom 3D printed guides were used to consistently and rigidly fix the femurs in optimal alignment.

Fractures in the femoral neck are a common occurrence in individuals with osteoporosis. Many mouse models have been developed to assess disease states and ...

Bioengineering

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

Understanding Tendon Impingement &#8211; Murine Explant Model for Studying Mechanobiology

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

Author Spotlight: Unraveling the Mechanobiology of Tendon Impingement &#8211; A Multiaxial Murine Hind Limb Explant Model 

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...

Translational Brain Mapping at the University of Rochester Medical Center: Preserving the Mind Through Personalized Brain Mapping

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