This study was supported by an NIH RO1 grant (1R01HD082302).

1. Preparation of the subject
<ol>
	<li>Prior to participation, have the participant complete a consent form and an MRI safety screening evaluation, the latter carried out by the neuroimaging technician at the scanning facility, to ensure that the participant does not have any known contraindications to being scanned (e.g., metal in their body, a history of claustrophobia, or pregnancy).</li>
	<li>Provide the participant with detailed instructions with regard to the experimental procedure.</li>
	<li>Have the subject li...

This protocol describes a novel, feasible procedure that allows investigators to accurately characterize the neural correlates associated with MT in individuals with PLP.
As previously mentioned, past studies have attempted to investigate the neural correlates associated with MT treatment by incorporating various techniques such as video recording, virtual reality, and prerecorded animations9,33,34. H...

PLP refers to the sensation of pain perceived within the area corresponding to the missing limb postamputation1,2. This condition is a significant chronic health care burden and can have a dramatic impact on an individual&#39;s quality of life3,4. It has been suggested that alterations in the brain structure and function play a fundamental role in the development and neuropathophysiology of PLP5,6. However, the underlying neural correlates of how pain symptoms develop and how they can be alleviated in response to treatment remain unknown. This lack of information is mostly due to technical challenges and limitations associated with performing a given therapeutic approach within the constraints of a neuroimaging environment such as MRI5,7,8.
Results from a number of studies attribute the development of PLP to maladaptive neuroplastic reorganization occurring within sensorimotor cortices, as well as in other areas of the brain. For example, it has been shown that following the amputation of a limb, there is a shift in the corresponding sensorimotor cortical representation of neighboring areas. As a result, neighboring areas apparently start invading the zones that used to correspond to the amputated limb9,10. In order to alleviate pain symptoms associated with PLP, treatments such as MT or motor imagery may be effective9,11,12. It is suggested that the alleviation of symptoms occurs putatively through the cross-modal re-establishment of afferent inputs, provided by the observation of mirror-reflected images from the nonaffected limb12,13,14,15,16,17. Through these images, participants are able to visualize the reflection of the opposite limb instead of the one that has been amputated, thus creating an illusion that both limbs remain. The illusion and immersive effects were previously studied by Diers et al. in healthy subjects in which a comparison of functional activation through functional MRI (fMRI) was evaluated after undergoing a task either with a common mirror box or virtual reality18. However, the neural correlates associated with the reversal of the maladaptive neuroplastic changes and the alleviation of symptoms remain poorly understood. Additionally, the underlying mechanism of PLP remains a topic of research as the clear underlying physiopathologic alteration behind the development of PLP is still incompletely elucidated while controversial findings have been revealed5,19. As stated above, multiple authors attribute the development of pain to deafferentation and cortical reorganization of the affected brain area (area of the amputated limb)6,7,8; however, opposite results were described by Makin and collaborators in which the presence of pain is associated with the preservation of brain structure and pain is attributed to a reduction interregional functional connectivity19. In view of these controversial and opposite findings, we believe that the novel approach presented here will bring additional relevant information to the study of PLP and will allow scientists to evaluate the effects of MT in a live environment with the degree of brain activation while comparing them with the levels of pain assessed in our full protocol19.
Previous literature on this topic has shown that MT is one of the most appropriate behavioral therapies for the treatment of PLP due to its easy implementation and low costs12. In fact, previous studies of this technique have shown evidence of a reversal of maladaptive changes within the primary sensorimotor cortex in amputees with PLP8,20,21. Even though MT is perhaps one the most inexpensive and most effective approach to treat PLP12,22,23,24, more studies are needed to confirm these effects since some patients do not respond to this type of treatment8 and there is a lack of larger randomized clinical trials that provide high-evidence-based results25.
One of the hypotheses by which MT can reduce PLP is related to the fact that the mirror image of the not-amputated body part helps to reorganize and integrate the mismatch between proprioception and visual feedback26. The underlying mechanisms of MT could be associated with the reversion of the maladaptive mapping of somatosensory8,27,28.
For MT, subjects are required to perform several motor and sensory tasks using their intact limb (e.g., flexion and extension) while observing this effect in a mirror located in the midline of the participant&#39;s body, thereby creating a vivid and precise representation of movement within the area of the amputated limb29.
To further develop the scientific understanding of the pathophysiology aspects involved in PLP, it is crucial to better characterize the underlying neuroplastic changes that result from limb amputations, as well as the improvement of pain symptoms provided by MT. In this regard, neuroimaging techniques, such as fMRI, have emerged as powerful tools to help elucidate the pathophysiologic mechanisms associated with cortical reorganization and provide clues toward optimizing the rehabilitation of individuals with PLP in the clinical context30,31. Furthermore, the high spatial resolution afforded by fMRI (as compared to electroencephalography, for example) allows for more accurate mapping of brain responses, such as finger and digit representations, in the sensorimotor cortex along with other regions of the brain32.
To date, the neurophysiology associated with MT remains elusive due in large part to the challenges of carrying out the procedure within the scanner environment (i.e., it is difficult for an individual to perform the therapy while lying in the scanner). Here, we describe a method that allows for an individual to observe their own leg movement in real-time while lying supine within the narrow confines of the scanner bore. An accurate recreation of the vivid and immersive sensation elicited by the therapy can be recreated using a video camera that captures real-time images of the moving leg, and a system of mirrors and a monitor that can be viewed directly by the study participant.
Past studies have attempted to incorporate techniques such as video recording, virtual reality, and prerecorded animations as means to present the visual stimulus and circumvent these technical challenges9,16,33,34. Yet, these techniques have been limited in their effectiveness35,36,37,38,39. In the particular case of using a prerecorded video, there is an often poor synchronization between participants&#39; movements and the ones provided by the video, as well as a lack of timing accuracy, which leads to a poor realistic impression that the individual&#39;s own leg is moving. In order to improve this sense of sensorimotor immersion, other techniques, such as virtual reality and digitized animations, have been attempted. Yet, they have failed to generate visually convincing sensations due to a low image resolution, a limited field of view, unrealistic or nonnatural human-like motions, and presence of motion lag (i.e., desynchronization of movement). Additionally, the lack of an accurate modeling combined with the poor control over other features, such as the effects of friction, momentum, and gravity, hinders the perception of a vivid and immersive feel40. Therefore, for amputees, it is worth to explore strategies to ensure that subjects are engaged in the cognitive task (observation) and immersive on the illusion of amputated limb movement. Finally, the required resources for developing and implementing these complex strategies may be time-consuming and/or cost prohibitive.
We describe a new approach that we believe creates a realistic and vivid sense of immersion whereby the participant can see a live and real-time video of a projected image of their own limb while they perform a session of the MT31. This approach is performed while the individual is lying in the scanner bore and is without substantial costs or extensive technical development.
This protocol is part of a National Institutes of Health (NIH) Research Project Grant (RO1)-sponsored clinical trial that evaluates the effects of the combination of a neuromodulatory technique, namely transcranial direct current stimulation (tDCS), with a behavioral therapy (mirror therapy) in order to relieve phantom limb pain31. We evaluate changes in the visual analog scale (VAS) for pain at baseline, prior, and after each intervention session. fMRI is used as a neurophysiologic tool in order to evaluate structural changes in brain function and its correlation with the relief of PLP. Therefore, an initial fMRI is obtained in order to have a baseline map of the structural organization of the participant&#39;s brain, which will either show that there is cortical maladaptive reorganization5,6,8,11,13,14,18,28 or that there is not19; in the same way, the scientist can observe what areas are activated at baseline with the task of MT in order to understand the areas&#39; activation response to the MT; lastly, it is possible to obtain a second fMRI postintervention to see if changes (modulation) have been generated in the cortical reorganization after the combined therapy with tDCS and MT and to analyze if those changes are correlated or associated with the degree of pain change. Therefore, this protocol allows scientists to evaluate structural reorganization changes in patients with PLPs during MT and also helps them to understand if these changes seen in fMRI are associated with changes in PLP, therefore providing additional details on how MT affects brain structural and functional activity to modify phantom pain.

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:137px;">Scanner</td>
			<td style="width:135px;">Phillips</td>
			<td style="width:129px;">NA</td>
			<td style="width:285px;">3 Tesla Philips Acheiva MRI scanner</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:137px;">Camera</td>
			<td style="width:135px;">Logitech</td>
			<td style="width:129px;">NA</td>
			<td style="width:285px;">HD Pro Webcam C910</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:137px;">Monitor</td>
			<td style="width:135px;">Cambridge Research Systems</td>
			<td style="width:129px;">NA</td>
			<td style="width:285px;">&#160;3D BOLD screen for MRI</td>
		</tr>
		<tr height="122">
			<td height="122" style="height:123px;width:137px;">Mirror</td>
			<td style="width:135px;">TAP Plastics</td>
			<td style="width:129px;">99999</td>
			<td style="width:285px;">Mirrored Acrylic Sheets (Cut&#173;to&#173;Size) &#173; Clear 1/8 (.118)&#34; Thick, 10&#34; Wide, 40&#34; Long</td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;width:137px;">Mirror stand</td>
			<td style="width:135px;"></td>
			<td style="width:129px;">NA</td>
			<td style="width:285px;">Mirror stand was built by the co-investigators from a rectangular piece of wood</td>
		</tr>
		<tr height="90">
			<td height="112" rowspan="2" style="height:113px;width:137px;">Headphones</td>
			<td rowspan="2" style="width:135px;">Westone Sensimetrics</td>
			<td rowspan="2" style="width:129px;">PN 79245</td>
			<td style="width:285px;">Replacement comply foam tips for universal-fit earphones. Canal size: Standard 6 pieces/ 3 pair&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:285px;">MR compatible in ear headphones</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">MRI Scanner</td>
			<td>Phillips</td>
			<td>3.0 T Philips Achieva System&#160;</td>
			<td></td>
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real-time video projection in an mri for characterization of neural correlates associated with mirror therapy for phantom limb pain

Mirror therapy (MT) has been proposed as an effective rehabilitative strategy to alleviate pain symptoms in amputees with phantom limb pain (PLP). However, establishing the neural correlates associated with MT therapy have been challenging given that it is difficult to administer the therapy effectively within a magnetic resonance imaging (MRI) scanner environment. To characterize the functional organization of cortical regions associated with this rehabilitative strategy, we have developed a combined behavioral and functional neuroimaging protocol that can be applied in participants with a leg amputation. This novel approach allows participants to undergo MT within the MRI scanner environment by viewing real-time video images captured by a camera. The images are viewed by the participant through a system of mirrors and a monitor that the participant views while lying on the scanner bed. In this manner, functional changes in cortical areas of interest (e.g., sensorimotor cortex) can be characterized in response to the direct application of MT.

Generating the sensation associated with MT using real-time video projection is feasible. Participants have subjectively reported that the video image perceived is life-like and the sensation is immersive.
Furthermore, the patterns of cortical activation associated with MT (i.e., movement of the leg and viewing the projected mirror image) in the scanner environment are robust. In a pilot study, the cortical responses to MT were ...

Watch this Scientific Journal Video about Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain at JoVE.com

Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain

We present a novel combined behavioral and neuroimaging protocol employing real-time video projection for the purpose of characterizing the neural correlates associated with mirror therapy within the magnetic resonance imaging scanner environment in leg amputee subjects with phantom limb pain.

Mirror therapy (MT) has been proposed as an effective rehabilitative strategy to alleviate pain symptoms in amputees with phantom limb pain (PLP). ...

The overall goal of this procedure is to more accurate characterize the neural correlates of mirror therapy in phantom limb pain patients, that is, PLP patients. This is accomplished by the following steps. Step one.Ensure that the participant does not have any known contraindications to MRI scanning and provide a pre-recorded audio to make sure that they are able to understand and follow the instructions provided during the scanning procedure. Step two. Position the patient as comfortably as possible in the scanner bed, where he or she should be lying supine with a single-piece MRI-compatible horizontal mirror between his or her legs.This mirror should be supported by a triangular stand to avoid contact with any part of the patient's body. Step three. Place an MRI-compatible digital camera on an adjustable tripod stand near the patient's intact leg to provide real-time video transmission.Step four, the final step. Begin with an anatomical MRI and adjust the machine settings to each patient and then, while the functional MRI scan is being performed, play the recording to the patient, instructing him or her to complete the specific behavioral tasks. Ultimately, the mirror attached to the MRI coil will allow patients to watch the mirrored leg movements in real time without moving their heads.For this protocol, you will need the following items. An MRI scanner, two MRI-compatible mirrors, a large one to place between the patient's legs, as well as a small one to place on the head coil. Additionally, you'll need sandbags, an MRI-compatible digital camera, a tripod for the camera, a computer-controlled system, and a monitor to place in the back of the scanner bore.Before proceeding to the MRI, it is critical to make sure the patient has no known contraindications to MRI scanning, for example, metal implants, aneurysm clips, or severe claustrophobia. Initially, you will explain to the patients exactly what they should expect during the experimental procedure. The patients will then listen to a recording with instructions to follow during the scan.Mirror.Patients may first practice during a mock scan to become familiar with the tasks as well as the scanner environment. The mock scanner is similar in every way to the real MRI scanner but without the active magnet. Before entering the scanner room, patients must remove their prostheses as well as any metal objects they might be wearing on their heads or bodies, for example, watches or jewelry.The MRI technician will make sure the patients have no metal that might put them at risk. All patients are transported to the scanner room using an MRI-safe wheelchair to avoid falling out. After that, the patients will transfer themselves to the MRI scanner bed.After the patient is lying comfortably in a supine manner on the scanner bed, a single-piece MRI-compatible horizontal mirror is placed between his or her legs. An adjustable arm is then positioned to point the camera at the mirrored leg. The large mirror is positioned between the legs at an angle of about 45 degrees, depending on the patient's height and amputation level.The goal is to cover the stump and make it invisible to the video camera. Sandbags are used to keep the mirror at the correct angle. A smaller mirror is positioned on the head coil, angled at 45 degrees at eye level.This mirror allows the patient to visualize the mirrored leg image directly, without moving their head while lying completely inside the scanner bore. An MRI-compatible digital camera is mounted on a tripod stand near the intact leg. This camera will transmit real-time video images of the mirrored leg movements to a computer control system that then projects the video to a monitor near the patient's head so he or she can view the mirrored leg movements.The patient will undergo a four-minute anatomical scan first followed by four runs of functional acquisitions while he or she performs the tasks. Each run lasts six minutes. During the scans, the patient wears sound-isolating MRI-compliant headphones which emit a series of auditory cues, instructing the patient to perform the given behavioral task.The following commands are used. One, leg, two, mirror, and three, rest. Additionally, the investigator says start and end at the beginning and end of the experimental run.The patient has already been instructed on hearing the word leg to follow the tapping sound presented in the audio. With the eyes closed, he or she will tap the foot at a rate of one tap per two seconds for a total of 10 taps in 20 seconds.Leg. On hearing the second command, mirror, the patient has to continue tapping his or her foot at the same rate, this time, while looking at the display showing the mirrored image of the two legs.Again, this would be at a rate of 10 taps in 20 seconds.Mirror. On hearing the third command, rest, the patient should stop moving his or her foot and lie motionless with both eyes closed.Rest. Data is collected in a single session for each patient and the entire scanning procedure lasts approximately 30 minutes.The investigators take note of any unwanted movements. Between the runs, they can ask the patients to keep the right pace and do the correct movements. After the procedure is finalized, the data is transferred to an encrypted flash drive and stored in a secure location in the facility.A longitudinal analysis design is used, comparing baseline and post-treatment data. The FSL software package and processing stream will be applied. Volumes with motion above 0.9 millimeters in any direction are identified with FSL's motion outlier detection processing stream and mathematically scrubbed from the final analysis.If more than 25%of the volumes are designated for removal, the whole acquisition is excluded from the total data set. A region of interest ROI analysis is used. The primary ROI is defined structurally using Freesurfer's Desikan Atlas of the primary sensory motor cortex and refined with a subject-specific functional activation during the leg versus stress condition at the baseline scan.This ROI is then reflected on the homologous area of the other hemisphere, that is, ipsilateral primary sensory motor representation of the intact lower limb. The secondary ROI is the entire bilateral occipital visual cortex as defined by the anatomical Desikan Atlas. Patients reported that the experience is immersive and the video image is life-like.Therefore, this real-time video projection process can generate the sensations associated with conventional mirror therapy. We expect that the leg condition, that is, the foot-tapping task, will lead to robust activation of the sensory motor cortex representing the intact leg compared to the rest condition. However, we also expect to see a less-pronounced activation of the sensory motor leg area representing the amputated leg.The mirror condition also shows robust contralateral as well as some ipsilateral activation of the cortical leg sensory motor area compared to the rest condition. Additionally, robust cortical activation is seen posteriorly in the visual cortical areas associated with viewing the mirrored leg. The activation pattern is described to represent the baseline condition, that is, prior to beginning therapy.These initial responses serve to define regions of interest, ROIs, and allow for comparisons after the therapeutic protocol is completed in each individual. After watching this video, you should have an adequate and sufficient understanding of the necessary steps required to set up all the equipments to perform your therapy inside the MRI scanner. This protocol describes a novel physical procedure that allows investigators to more accurately characterize the neural correlates associated with mirror therapy in individuals with phantom limb pain.We could answer additional questions regarding brain organization after a limb amputation following the steps of this protocol using other neurophysiological measurements or imaging techniques. A challenge associated with this approach is the risk of generating excessive head motion artifacts, given that the leg must be moved repeatedly inside the scanner. Excessive head motion can compromise image data quality.In this regard, it's important to plan ahead and implement a variety of strategies to mitigate this possibility. These include training the participant in a mock scanner to carry out the task without excessively moving their head, making sure that the head is secure yet comfortably restrained, and implementing motion detection correction strategies during the acquisition and data analysis phases respectively. Given the method to implementing the experimental set-up is relatively simple, this approach may allow the evaluation of the mirror therapy effects not only in limb amputees but also in other conditions, such as stroke or spinal cord injury where mirror therapy is already commonly used in clinical practice.

Real-time Video Projection in an MRI for Characterization of Neural Correlates Associated with Mirror Therapy for Phantom Limb Pain

Abstract