This work was supported by the following grants (DaSilva AF): National Institute of Health - National Institute of Neurological Disorders and Stroke - K23 NS062946, Dana Foundation's Brain and Immuno-Imaging Award, and the Migraine Research Foundation Research Grant Award. The authors acknowledge the PET Center Nuclear Medicine Technologists (Jill M. Rothley, Edward J. McKenna, Andrew R. Weeden, Paul Kison, and Caitlin Hendricks) and the personnel of Functional MRI Laboratory (Scott Peltier and Keith Newnham). Dr. Alexandre DaSilva, the principal investigator, had full access to all the data in the study, and takes responsibility for the integrity of the data and the accuracy of the data analysis. The authors declare no conflicts of interest related to this study.

The study should be approved by the local Institutional Review Board, and by the Radioactive Drug Research Committee. The research subject gives written informed consent to participate in the study. The protocol is divided into three chronological steps:
<ol>
	<li>MRI session</li>
	<li>PET session during the ictal phase (headache) of migraine</li>
	<li>PET session during the interictal phase (no headache) of migraine</li>
</ol>
The patient is responsible for filling out a headache diary, and for confirming with the research group the occurrence of a migraine attack on the day of imaging session. Both, interictal and ictal, PET scans should be scheduled during separate mid-late follicular phases of the patient (5 to 10 days after the first day of menstrual bleeding), which in this case was tracked and calculated in advance by a gynecologist with expertise in the field (Y.R.S.).
1. MRI Session
<ol>
	<li>Preparing for the Experiment
	<ol>
		<li>Prior to preparing the subject for the scan, it is necessary to follow proper safety instructions due to the magnetic field of the MR scanner. All study personnel must be metal-free before entering the MR procedure room.</li>
		<li>Provide a copy of the Informed Consent Form, previously signed by the research volunteer during initial screening, to the MR technologist.</li>
	</ol>
	</li>
	<li>Preparing the Subject for the Scan
	<ol>
		<li>On the day of the MRI, ask the research subject to fill out a MRI safety screening form. This form is required for any MRI taken at the University of Michigan &#8211; fMRI lab. The questionnaire reinforces that the subject is also metal-free and does not have a medical condition that requires careful and special consideration (e.g., metallic foreign fragments, implanted mechanical/electrical device).</li>
		<li>Reassure that the participant understands the MR procedure, risks and benefits.</li>
		<li>Deliver the completed screening form to the MR technologist who will be assisting in the procedure.</li>
	</ol>
	</li>
	<li>Acquire a T1-weighted anatomical MRI scan during an interictal phase for the patient on a 3 Tesla scanner.
	<ol>
		<li>Use the following sequence parameters for the MRI acquisition:
		<ul>
			<li>A. Axial spoiled-gradient recalled (SPGR) 3D acquisition</li>
			<li>B. Bandwith = 15.63</li>
			<li>C. Repetition time [TR] = 9.2 msec</li>
			<li>D. Echo time [TE] = 1.9 msec</li>
			<li>E. Inversion recovery preparation 500 msec</li>
			<li>F. Flip angle = 15&#176;</li>
			<li>G. 25/26 FOV</li>
			<li>H. Number of Excitations [NEX] = 1</li>
			<li>I. 144 contiguous slices</li>
			<li>J. 1.0 mm slice thickness</li>
			<li>K. 256 x 256 matrix</li>
		</ul>
		</li>
	</ol>
	</li>
</ol>
2. Ictal PET Session
<ol>
	<li>Preparing for the Experiment
	<ol>
		<li>Prior to confirming the scan at the University Hospital, contact the subject to verify in which phase of the menstrual cycle she will be on the day of the scan. It is recommended to perform a PET scan during mid-late follicular phase (5 to 10 days following menstrual bleeding onset).</li>
		<li>Submit a request to the hospital to produce [11C]carfentanil, a short-life radiotracer with a selective affinity for &#181;-opioid receptors, using a cyclotron in the vicinity of the scan. The tracer must be produced 2 hr before the scan.</li>
		<li>On the day of the potential ictal PET scan, contact the subject 2&#160;hr&#160;before the appointment to confirm the presence of a spontaneous migraine attack. If a migraine attack is present, validate the migraine diagnosis following the International Classification of Headache Disorders. After diagnosis, assure that the participant is able to safely get to the hospital to undergo the scan. Provide transportation if the subject is not comfortable driving or if no designated driver is available.</li>
	</ol>
	</li>
	<li>Preparing the Subject for the Scan
	<ol>
		<li>When the participant arrives in the hospital, escort her to the PET suite for revalidation of diagnosis based on the International Classification of Headache Disorders. Prior to the scan, perform a urine drug test to confirm that the subject did not intake any substance that could interact with the tracer [11C]carfentanil, followed by an urine pregnancy test.</li>
		<li>Reaffirm that the participant understands the PET procedure, risks, and benefits.</li>
		<li>Deliver a copy of the Informed Consent Form, previously signed by the research volunteer during the initial screening, to the Nuclear Medicine technologist.</li>
		<li>Following the guidance of the Nuclear Medicine technologist, help the subject settle into the scanner.</li>
		<li>Have the subject undergo 1&#160;90 min PET scan using a Siemens HR+ scanner in 3D mode (reconstructed images have a full-width at half maximum (FWHM) resolution of ~5.5 mm-in-plane and 5.0 mm axially).</li>
		<li>For each [11C]carfentanil dose (555 MBq &#8804; 0.03 &#956;g/kg), administer fifty percent as a bolus with the remainder continuously infused over the course of the scan to achieve steady-state tracer levels approximately 35 min&#160;after tracer administration.</li>
		<li>Interictal PET session.</li>
		<li>Repeat Steps 2.2 - 2.6 during non-headache phase.</li>
	</ol>
	</li>
</ol>
3. PET Data Reconstruction
<ol>
	<li>Reconstruct PET images using interactive algorithms into a 128 x 128 pixel matrix in a 28.8 cm diameter field of view (FOV).</li>
	<li>Acquire 21&#160;image frames and co-register to one another to correct for motion during the scan.</li>
	<li>Obtain a 6 min transmission (68Ge source) scan prior to the PET scan for the purposes of attenuation correction.</li>
	<li>Convert the dynamic image data for each scan on a voxel-by-voxel basis into two sets of parametric images (10 - 40 min):
	<ol>
		<li>Use a tracer transport measure (K1 ratio) for coregistration and normalization procedures; and</li>
		<li>Use a receptor-related measure, BPND, proportional to Bmax (receptor concentration) to divide by Kd (receptor affinity).</li>
	</ol>
	</li>
	<li>Estimate these measures by using the reference region-based Logan graphical analysis with the occipital cortex as the reference region12.</li>
</ol>
4. PET Data Analysis
NOTE: Anatomically standardize images into template space using Statistical Parametric Mapping (SPM8) software following the sequence below.
<ol>
	<li>Co-register the MR scan and K1 scans.</li>
	<li>Normalize the MR scan to the Montreal Neurologic Institute (MNI) template brain using DARTEL.</li>
	<li>Apply the resulting deformation matrix to the PET images.</li>
	<li>Verify co-registration and normalization accuracy by comparing the transformed MR and PET images to the MNI atlas template.</li>
	<li>Region of Interest (ROI) analysis.</li>
</ol>
Examine the activity of several bilateral regions that are engaged during the processing of pain, including:
<ul>
	<li>A. Anterior/medial/posterior cingulate</li>
	<li>B. Insula</li>
	<li>C. Hippocampus</li>
	<li>D. Amygdala</li>
	<li>E. Caudate head/body</li>
	<li>F. Nucleus accumbens</li>
	<li>G. Putamen</li>
	<li>H. Lateral/medial globus pallidus</li>
	<li>I. Thalamus nuclei (ventral anterior, ventral posterior lateral/medial, lateral posterior, midline, lateral/medial dorsal)</li>
	<li>J. Periaqueductal gray matter (PAG)</li>
</ul>
<ol>
	<li>Define MarsBaR (in standardized space for each of these regions, with the exception of the PAG. Generate the PAG ROI by placing a 3 mm sphere at coordinates: right: 4, -28, -6; and left: -2, -28, -6. This PAG location was previously shown to have diffusional and connectivity alterations in migraine patients as compared to healthy controls13,14. 
	NOTE: Test-retest studies with [11C]carfentanil show reproducibility of BPND measures of well less than 10%, with most cortical regions being 3 - 5%15. The largest coefficients of variation (CoV = Std.Dev./Mean) are typically observed in the regions with the lowest binding, however, even in the cortical regions with lowest binding BPND CoV&#8217;s were ~0.5. Hence, percentage changes in ROI &#181;OR BPND between scans are only considered as significant when above 10%.</li>
</ol>
5. 3D-Neuronavigation
<ol>
	<li>Preparing for an 3D-IIN Experience
	<ol>
		<li>Organize the data provided in NIfTI volumetric data format as a stack of images with density and activation levels defined as 16 bit.</li>
		<li>Wear active LCD shutter glasses to enable the time sequential stereoscopic 3D effect. The shutter glasses operate through the blocking of one eye while the image for the other eye is shown on screen. The process then repeats for the other eye. This shuttering effect occurs at 110 Hz.</li>
		<li>Use a joystick for interactions with the simulation after being instructed on its use.</li>
		<li>Outfit the shutter glasses and joystick with reflective markers to enable precise 6DOF tracking of the objects in space via a Vicon motion capture system.</li>
	</ol>
	</li>
	<li>Display Aubject Activation Data
	<ol>
		<li>Use XML configuration files to define color mappings of density and activation levels, which are loaded when the application begins and shared with each computer in the cluster.</li>
		<li>Acquire 3-dimensional volumetric cells from the provided NIfTIdata set by way of Jugular&#8217;s internal loading functions and the &#8220;Niftilib&#8221; open source software library.</li>
		<li>Share resulting volumetric cells with each computer in the cluster to improve speed.</li>
		<li>Interpret Volumetric cells by an OpenGL shader (GLSL) that conducts ray marching and the displaying of voxels with varying colors and transparencies defined by previously shared color mapping XML configuration files.</li>
	</ol>
	</li>
	<li>Obtain the location through the Vicon system and use this to update drawn perspectives of the volumetric data on each screen.</li>
	<li>Record interactions and use them to dynamically adjust and cut planes through the data in order to navigate in the virtual space.</li>
</ol>
6. 3D-Immersive &#38; Interactive Neuronavigation (3D-IIN)
<ol>
	<li>Store subject activation data in the NIfTI data format, a volumetric data type that is interpreted using the Niftilib library.</li>
	<li>Obtain interactions and the location through a Vicon tracking system, a joystick device, and gestural input. Use this information to ensure the displayed image represents the correct vantage point, allowing for real-time exploration of the data set, and enable dynamic control for up to 3 arbitrary cutting planes using familiar movements and control schemes (Figure 1).</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migraine:+epidemiology,+impact,+and+risk+factors+for+progression.">Migraine: epidemiology, impact, and risk factors for progression.</a> Headache. , (2005).</li><li>Hu, X. H., Markson, L. E., Lipton, R. B., Stewart, W. F., Berger, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+migraine+in+the+United+States:+disability+and+economic+costs.+Arch+Intern+Med.">Burden of migraine in the United States: disability and economic costs. Arch Intern Med.</a> Arch Intern Med. 159, 813-818 (1999).</li><li>Lipton, R. 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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interictal+alterations+of+the+trigeminal+somatosensory+pathway+and+periaqueductal+gray+matter+in+migraine.">Interictal alterations of the trigeminal somatosensory pathway and periaqueductal gray matter in migraine.</a> Neuroreport. 18, 301-305 (2007).</li><li>Hirvonen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+central+mu-opioid+receptor+binding+in+vivo+with+PET+and+[11C]carfentanil:+a+test-retest+study+in+healthy+subjects.">Measurement of central mu-opioid receptor binding in vivo with PET and [11C]carfentanil: a test-retest study in healthy subjects.</a> European journal of nuclear medicine and molecular imaging. 36, 275-286 (2009).</li><li>Cao, Y., Aurora, S. K., Nagesh, V., Patel, S. C., Welch, K. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Painful+heat+reveals+hyperexcitability+of+the+temporal+pole+in+interictal+and+ictal+migraine+States.">Painful heat reveals hyperexcitability of the temporal pole in interictal and ictal migraine States.</a> Cereb Cortex. 21, 435-448 (2011).</li><li>Burstein, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalamic+sensitization+transforms+localized+pain+into+widespread+allodynia.">Thalamic sensitization transforms localized pain into widespread allodynia.</a> Ann Neurol. 68, 81-91 (2010).</li><li>Denuelle, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PET+study+of+photophobia+during+spontaneous+migraine+attacks.">A PET study of photophobia during spontaneous migraine attacks.</a> Neurology. 76, 213-218 (2011).</li><li>Denuelle, M., Fabre, N., Payoux, P., Chollet, F., Geraud, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+activation+in+spontaneous+migraine+attacks.">Hypothalamic activation in spontaneous migraine attacks.</a> Headache. 47, 1418-1426 (2007).</li><li>Weiller, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+stem+activation+in+spontaneous+human+migraine+attacks.">Brain stem activation in spontaneous human migraine attacks.</a> Nat Med. 1, 658-660 (1995).</li><li>Williamson, D. 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The authors have nothing to disclose.

In this case report, actual migraine headache neuroimaging data was explored for the first time, in a fully immersive virtual 3D reality, which demonstrated a decrease in &#181;-opioid receptor availability (&#181;OR BPND). Reductions in &#181;OR BPND imply that there is a higher occupancy and/or a loss of &#181;-opioid receptors in the central nervous system. Acute reductions in &#181;OR BPND in pain-matrix regions during the ictal scan as compared to the interictal scan, are expected to occur as a consequence of the release of endogenous opioids interacting with &#181;ORs as a regulatory response to the ongoing pain, making less &#181;ORs accessible to the radiotracer.
The novelty of our ictal migraine neuroimaging study lies in the new 3D neuronavigation approach to investigate a patient&#8217;s actual brain data in virtual reality. PET radiotracer technology was used to measure changes in &#181;ORs availability with [11C]carfentanil. When examined during the headache event, the brains of migraineurs are usually scanned following an attack trigger (e.g., glyceryl trinitrate, photostimulation)16,17, or under the technical demand of a specific evoked stimulus (e.g., pain, brush, light, and odor)18-20. All those studies corroborate the knowledge that the disorder is associated with cortical and subcortical hyperexcitability during the headache phase. However, such a plethora of stimuli in the neuroimaging protocols introduces multiple factors that cloud our understanding of the sole impact of an acute migraine attack on the central nervous system. From the few previous functional studies without the presence of exogenous stimulation, there is indication of increased regional cerebral flow in areas such as the cingulate cortex, hypothalamus, and brainstem21, which can persist even after acute therapy22. Hitherto, the neuroimaging technologies applied have not allowed for the molecular characterization of neurotransmitter/receptor processes involved in the migraine attack, such as the endogenous &#181;-opioid mechanism, one of most important analgesic resources in the brain. Moreover, our method allowed these processes to be explored using 3D neuronavigation in a virtual environment.
The descending pain modulatory system is a complex network that regulates pain processing to a great extent via &#956;-opioid receptors throughout the brain, including spinal to supra-spinal areas. These areas are known to be involved in endogenous anti-nociception, stress-induced analgesia, and in the action of opioid drugs commonly used for chronic pain and migraine treatment. In fact, the dural neurogenic vasodilation associated with migraine can be inhibited by morphine and subsequently reversed by the opioid antagonist naloxone, indicating that the effects of morphine on neurogenic inflammation are mediated specifically via activation of &#181;-opioid receptors23. Interestingly, the magnitude of endogenous opioid/&#181;ORs regional activations in humans is related to the individual&#8217;s capacity to suppress sensory and affective elements of the pain experience24.
In our study, the brain regions that showed reductions in &#181;-opioid receptor availability during the ictal phase are responsible for both elements of the migraine headache experience and its modulation. They are the ACC, thalamus, basal ganglia (e.g., NAcc), hippocampus, and the PAG. In addition to sensitization due to abnormal trigeminal afferent traffic, one solid hypothesis for migraine pathophysiology is the dysfunction of the modulatory system. In this case, projections from/to brainstem structures, such as the PAG, where there is a high expression of opioid receptors, would inefficiently produce their anti-nociceptive effect on ascending sensory neurons. In addition, other higher cortical structures participate in this faulty pain-modulatory mechanism in migraine. A recent interictal resting-state study reported changes in the connectivity of migraineurs versus healthy controls in the ventrolateral PAG and most of the (sub)cortical structures in the modulatory pain system and correlated these with the frequency of the headache attacks13. The regions with connectivity changes found in this study are the same as those with changes in &#181;OR BPND found in our own study. The same PAG location was originally reported as encompassing microstructural neuroplasticity in migraineurs14, and here had a considerable decrease in &#181;OR BPND during the attack.
Further studies with larger cohorts are necessary to confirm and extend the findings presented in this case report. For instance, it is currently unknown why the system does not properly respond to the long-term use of exogenous opioids frequently prescribed in migraine clinics. Nonetheless, our study provides important mechanistic information, on the impact of a migraine headache in the &#181; -opioid system, and uses a novel 3D immersive and interactive neuronavigation (3D-IIN) approach for the first time. In the future, this exploratory 3D method could provide a much more immersive and interactive perspective for examining the brains of patients in research and the clinic.

Migraine is a chronic trigeminal pain disorder that affects nearly 16% of women and 6% of men in the United States and worldwide1-3. Repetitive migraine headache attacks have an impact on a large part of the patient&#8217;s existence, impairing quality of life and performance, costing billions of dollars in missed school/work days and healthcare utilization4. During the debilitating headache attacks, its sufferers have a marked increased sensitivity to noxious (hyperalgesia) and even nonnoxious stimuli (allodynia)5.
The &#181;-opioid neurotransmitter system is one of the principal endogenous pain modulatory mechanisms in our brain. It is involved in the regulation of experimental and clinical pain perception, as well as in the analgesic action of opioid drugs6-9&#160;which have been associated with the chronification of migraine attacks10. Recent advances in Positron Emission Tomography (PET) molecular imaging allow for the study of important molecular mechanisms in the brain of chronic pain patients in vivo11. In this study, despite the challenging logistics of synchronizing the aleatory and debilitating nature of the episodic attacks with the complexity of PET/radiotracer session setup, 3D neuronavigation was used for the first time to investigate &#181;OR availability in key pain-matrix regions of a patient&#8217;s brain during a spontaneous migraine headache.
Case Presentation
A 36 year-old Asian female was enrolled in the study. She presented with a 10 year history of migraine with visual aura. Right-sided migraine headaches occurred an average of 12 days per month, with moderate to severe pain intensity that would usually last for 72 hr (if untreated or unsuccessfully treated). There was an increased frequency of headache attacks around her menstrual cycle, which had a regular pattern throughout the study. Associated symptoms included: nausea, vomiting, photophobia, and phonophobia. During the regular headache attacks she did not present any autonomic symptoms. As treatment, she was managing her symptoms with pharmacological abortive therapy only, which was based on non-steroidal anti-inflammatory drugs, and there were no indications of medication overuse or opioid intake. The clinical examination during the screening visit was unremarkable and without abnormalities, and a review of systems was within normal limits. She was single with no children, and was not using contraceptive medication.

3d-neuronavigation in vivo through a patient's brain during a spontaneous migraine headache

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration of specific neural processes in the central nervous system. However, information is lacking on the molecular impact of these changes, especially on the endogenous opioid system during migraine headaches, and neuronavigation through these changes has never been done. This study aimed to investigate, using a novel 3D immersive and interactive neuronavigation (3D-IIN) approach, the endogenous &#181;-opioid transmission in the brain during a migraine headache attack in vivo. This is arguably one of the most central neuromechanisms associated with pain regulation, affecting multiple elements of the pain experience and analgesia. A 36 year-old female, who has been suffering with migraine for 10 years, was scanned in the typical headache (ictal) and nonheadache (interictal) migraine phases using Positron Emission Tomography (PET) with the selective radiotracer [11C]carfentanil, which allowed us to measure &#181;-opioid receptor availability in the brain (non-displaceable binding potential - &#181;OR BPND). The short-life radiotracer was produced by a cyclotron and chemical synthesis apparatus on campus located in close proximity to the imaging facility. Both PET scans, interictal and ictal, were scheduled during separate mid-late follicular phases of the patient's menstrual cycle. During the ictal PET session her spontaneous headache attack reached severe intensity levels; progressing to nausea and vomiting at the end of the scan session. There were reductions in &#181;OR BPND in the pain-modulatory regions of the endogenous &#181;-opioid system during the ictal phase, including the cingulate cortex, nucleus accumbens (NAcc), thalamus (Thal), and periaqueductal gray matter (PAG); indicating that &#181;ORs were already occupied by endogenous opioids released in response to the ongoing pain. To our knowledge, this is the first time that changes in &#181;OR BPND during a migraine headache attack have been neuronavigated using a novel 3D approach. This method allows for interactive research and educational exploration of a migraine attack in an actual patient's neuroimaging dataset.

The patient presented to the hospital with a right temporal and occipital pulsating headache, with intensity of 6 on a 0-10 pain scale. She was having her typical migraine headache, however without aura. It had initiated upon awakening 5 hr before the (ictal) PET session, and she managed to tolerate it without any abortive pharmacotherapy. To her knowledge, the headache was not evoked by any triggering factor (e.g., alcohol, sleep deprivation). No autonomic symptoms were reported, but photophobia and phonophobia were present. Following initiation of the ictal PET session the headache intensity escalated, reaching severe levels (9 on a 0-10 pain scale) 60 min into the study; progressing to nausea and vomiting at the end of the scan session. Decrease in &#181;OR BPND was noticed in the brain of the patient during the spontaneous migraine headache (ictal phase), as compared to the baseline (interictal phase) (Figure 2). There were patent reductions in &#181;OR BPND in the main pain-matrix regions of the endogenous &#181;-opioid system, including the following thalamus nuclei: right lateral dorsal (11, -19, -16: 10.2%), right medial dorsal (6, -17, -8: 11.1%), right midline (8, -19, -16: 27%), and ventral anterior (9, -9, -12: 12.0%). In addition, changes were found in the right anterior (8, 35, 14: 13.7%) and left posterior cingulate cortex (-5, -44, 23: 11.8%), left caudate body (-11, 6, 15: 12.0%), medial globus pallidus (right: 16, -4, -3: 16.2%; left: -14, -4, -3: 22.6%), left nucleus accumbens (-9, -11, -7: 10.5%), and hippocampus (right: 30, -22, -14: 12.6%; left: -30, -22, -14: 11.5%). There was an increase in &#181;OR BPND only in the left amygdala (-23, -4, -19: 11.7%). In the brainstem, the significant ictal reduction in &#181;OR BPND extended from the rostral to caudal periaqueductal gray matter (PAG) (right: 4, -28, -6: 15.1%; left: -2, -28, -6: 14.6%) (Figure 3). However, the global hemispheric percentage changes in the &#181;OR BPND during the migraine attack were modest (right: 8.5%; left: 8.29%), indicating that the decreases in the &#181;OR BPND were specific to the pain-matrix structures in the brain.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50682/50682fig1highres.jpg" width="500" /> 
Figure 1.&#160;Full Virtual Reality 3D Data Navigation of a Migrainous Brain. For the first time actual migraine neuroimaging data was explored in a fully immersive 3D virtual reality, which includes unrestricted navigation through the data (by students, clinicians, and researchers) regarding availability of &#181;-opioid receptors (&#181;OR BPND) in the brain during the migraine headache attack in vivo.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50682/50682fig2highres.jpg" width="500" /> 
Figure 2.&#160;&#956;-Opioid Brain Profile of a Migraine Headache in vivo.&#160;The ictal phase (lower row) &#8211; headache phase - shows a decrease in &#956;-opioid receptor availability (&#181;OR BPND) in the pain-matrix regions (Threshold value, DV = 4.50). This result possibly represents an increase in endogenous &#956;-opioid release during the migraine attack, as a regulatory response to the ongoing severe headache. Key words: thalamus (Thal), nucleus accumbens (Nac), and anterior cingular cortex (ACC).
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/50682/50682fig3highres.jpg" width="500" /> 
Figure 3.&#160;Midbrain/Pons/Medulla &#956;-Opioid Receptor Availability During a Migraine Attack in vivo. The ictal phase (right column) &#8211; headache phase - shows a decrease in &#956;-opioid receptor availability along the periaqueductal gray matter (PAG) (Threshold value, DV = 4.50), as compared to the interictal phase (left column) &#8211; non-headache phase. Key words: PAG: r - rostral; m - medial; c - caudal.

Watch this Scientific Journal Video about 3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache at JoVE.com

3D-Neuronavigation In Vivo Through a Patient&#039;s Brain During a Spontaneous Migraine Headache

In this study, the authors report for the first time a novel 3D-Immersive &#38; Interactive Neuronavigation (3D-IIN) through the impact of a spontaneous migraine headache attack in the &#956;-opioid system of a patient's brain in vivo.

3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache

A growing body of research, generated primarily from MRI-based studies, shows that migraine appears to occur, and possibly endure, due to the alteration ...

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17.8K Views.  University of Michigan School of Dentistry. In this study, the authors report for the first time a novel 3D-Immersive & Interactive Neuronavigation (3D-IIN) through the impact of a spontaneous migraine headache attack in the μ-opioid system of a patient's brain in vivo.

Video: 3D-Neuronavigation In Vivo Through a Patient's Brain During a Spontaneous Migraine Headache

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive astrogliosis. Reactive astrocytes exert both inflammatory and protective effects that inhibit and promote, respectively, neural repair. The mechanisms underlying the diverse functional properties of reactive astrogliosis are not well understood. Achieving a greater understanding of these mechanisms is critical to developing therapeutic strategies to treat the injured CNS. Here we demonstrate a method to trigger reactive astrogliosis in the adult mouse forebrain using a forebrain stab lesion. This lesion model is simple, reliable, and requires only a stereotaxic device and a scalpel blade to produce the injury. The use of stab lesions as an injury model in the forebrain is well established and amenable to studies addressing a broad range of neuropathological outcomes, such as neuronal degeneration, neuroinflammation, and disruptions in the blood brain barrier (BBB). Thus, the forebrain stab injury model serves as a powerful tool that can be applied for a broad range of studies on the CNS response to trauma.

Triggering Reactive Gliosis In Vivo by a Forebrain Stab Injury

This article describes a detailed protocol to produce a forebrain stab injury in adult mice. The stab injury induces severe reactive gliosis and glial scar formation which can be subsequently examined by standard immunohistochemistry methods.

Following injury to the CNS, astrocytes undergo a broad range of biochemical, morphological, and molecular changes collectively referred to as reactive ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...