Name: a human-machine-interface integrating low-cost sensors with a neuromuscular electrical stimulation system for post-stroke balance rehabilitation
Uploaded: 2016-04-12T13:00:00
Description: Watch this Scientific Journal Video about A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation at JoVE.com

Research conducted within the context of the Joint targeted Program in Information and Communication Science and Technology - ICST, supported by CNRS, Inria, and DST, under CEFIPRA&#39;s umbrella. The authors would like to acknowledge the support of students, specifically Rahima Sidiboulenouar, Rishabh Sehgal, and Gorish Aggarwal, towards development of the experimental setup.

Note: The HMI software pipeline was developed based on freely available open-source software and off-the-shelf low-cost video game sensors (details available at: https://team.inria.fr/nphys4nrehab/software/ and https://github.com/NeuroPhys4NeuroRehab/JoVE). The HMI software pipeline is provided for data collection during a modified functional reach task (mFRT)21 in a VR based gaming platform for visuomotor balance therapy (VBT)8.
Figure 2a shows the diagn...

<ol>
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A simple-to-use, clinically valid low-cost tool for movement and balance therapy will be a paradigm shift for neurorehabilitation in a low-resource setting. It is likely to have a very high societal impact since neurological disorders like stroke will dramatically increase in future due to aging world population2. There is, therefore, a pressing need to leverage cyber physical systems where the ability to customize, monitor, and support neuro-rehabilitation at remote sites has recently become possible with the...

An episode of neurological dysfunction caused by focal cerebral, spinal, or retinal infarction is called stroke1. Stroke is a global health problem and fourth leading cause of disability worldwide1. In countries like India and China, the two most populous nations of the world, neurologic disability due to stroke is being labeled as hidden epidemic2. One of the most common medical complications after a stroke are falls with a reported incidence of up to 73% in the first year post-stroke3. The post-stroke fall is multifactorial and includes both spinal and supraspinal factors like balance and visuospatial neglect4. A review by Geurts and colleagues5 identified 1) multi-directionally impaired maximal weight shifting during bipedal standing, 2) slow speed, 3) directional imprecision, and 4) small amplitudes of single and cyclic sub-maximal frontal plane weight shifts as the balance factors for fall risk. The consequent impact on activities of daily living can be significant since prior works have shown that balance is associated with ambulatory ability and independence in gross motor function5,6. Moreover, Geurts and colleagues5 suggested that supraspinal multisensory integration (and muscle coordination7) in addition to muscle strength is critical for balance recovery which is lacking in current protocols. Towards multisensory integration, our hypothesis8 on volitionally driven non-invasive electrotherapy (NMES/SES) is that this adaptive behavior can be shaped and facilitated by modulating active perception of sensory inputs during NMES/SES-assisted movement of the affected limb such that the brain can incorporate this feedback into subsequent movement output by recruiting alternate motor pathways9, if needed.
To achieve volitionally driven NMES/SES-assisted balance training in a resource-poor setting, a low-cost human-machine-interface (HMI) was developed by leveraging available open-source software and recent advances in off-the-shelf video game sensor technology for visual-auditory biofeedback. NMES involves coordinated electrical stimulation of nerves and muscles that has been shown to improve muscle strength and reduce spasticity10. Also, SES involves stimulation of sensory nerves with electrical current to evoke sensations where preliminary published work11 showed that subsensory stimulation applied over the tibialis anterior muscles alone is effective in attenuating postural sway. Here, the HMI will make possible sensory-motor integration during interactive post-stroke balance therapy where volitionally-driven NMES/SES for the ankle muscles will act as a muscle amplifier (with NMES) as well as enhance afferent feedback (with SES) to assist healthy ankle strategies12,13,14 to maintain upright stance during postural sways. This is based on the hypothesis presented in Dutta et al.8 that an increased corticospinal excitability of relevant ankle muscles effected through non-invasive electrotherapy may lend to an improved supraspinal modulation of ankle stiffness. Indeed, prior work has shown that NMES/SES elicits lasting changes in corticospinal excitability, possibly as a result of co-activating motor and sensory fibers15,16. Moreover, Khaslavskaia and Sinkjaer17 showed in humans that concurrent motor cortical drive present at the time of NMES/SES enhanced motor cortical excitability. Therefore, volitionally-driven NMES/SES may induce short-term neuroplasticity in spinal reflexes (e.g., reciprocal Ia inhibition17) where corticospinal neurons that project via descending pathways to a given motoneuron pool can inhibit the antagonistic motoneuron pool via Ia-inhibitory interneurons in humans18, as shown in Figure 1, towards an operant conditioning paradigm (see Dutta et al.8).
<img alt="Figure 1" src="/files/ftp_upload/52394/52394fig1.jpg" /> 
Figure 1: The concept (details at Dutta et al.21) underlying interactive human machine interface (HMI) to drive the center of pressure (CoP) cursor to the cued target to improve ankle muscle coordination under volitionally driven neuromuscular electrical stimulation (NMES)-assisted visuomotor balance therapy. EEG: electroencephalography, MN: &#945;-motoneuron, IN: Ia-inhibitory interneuron, EMG: electromyogram, DRG: dorsal root ganglion. Reproduced from8 and37&#8203;.&#160;<a href="https://www.jove.com/files/ftp_upload/52394/52394fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The antero-posterior (A-P) displacements in center of mass (CoM) are performed by ankle plantarflexors (such as medial gastrocnemius and soleus muscles) and dorsiflexors (such as the anterior tibial muscle) while medio-lateral (M-L) displacements are performed by ankle invertors (such as the anterior tibial muscle) and evertors (such as peroneus longus and brevis muscles). Consequently, stroke-related ankle impairments including weakness of the ankle dorsiflexor muscles and increased spasticity of the ankle plantarflexor muscles lead to impaired postural control. Here, agility training programs6 can be leveraged in a virtual reality (VR) based gaming platform that challenge dynamic balance where tasks are progressively increased in difficulty which may be more effective than static stretching/weight-shifting exercise program in preventing falls6. For example, subjects can perform volitionally driven NMES/SES assisted A-P and M-L displacements during a dynamic visuomotor balance task where the difficulty can be progressively increased to ameliorate post-stroke ankle-specific control problems in weight shifting during bipedal standing. Towards volitionally driven NMES/SES assisted balance therapy in a resource-poor setting, we present a low-cost HMI for Mobile Brain/Body Imaging (MoBI)19, towards visual-auditory biofeedback which can also be used for data collection from low-cost sensors for offline data exploration in MoBILAB (see Ojeda et al.20).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>NMES stimulator</td>
			<td>Vivaltis, France</td>
			<td>PhenixUSBNeo</td>
			<td>NMES stimulator cum EMG sensor (Figure 2b)</td>
		</tr>
		<tr>
			<td>Balance Board</td>
			<td>Nintendo, USA</td>
			<td>Wii Balance Board</td>
			<td>Balance Board (Figure 2b)</td>
		</tr>
		<tr>
			<td>Motion Capture</td>
			<td>Microsoft, USA</td>
			<td>XBOX-360 Kinect</td>
			<td>Motion Capture (Figure 2b)</td>
		</tr>
		<tr>
			<td>Eye Tracker&#160;</td>
			<td>Eye Tribe</td>
			<td>The Eye Tribe</td>
			<td>SmartEye Tracker (Figure 2a)</td>
		</tr>
		<tr>
			<td>EEG Data Acquisition System</td>
			<td>Emotiv, Australia</td>
			<td>Emotiv Neuroheadset</td>
			<td>Wireless EEG headset (Figure 2b)</td>
		</tr>
		<tr>
			<td>EEG passive electrode</td>
			<td>Olimex</td>
			<td>EEG-PE</td>
			<td>EEG passive electrode for EOG and references (6 in number)(Figure 2b)</td>
		</tr>
		<tr>
			<td>EEG active electrode</td>
			<td>Olimex</td>
			<td>EEG-AE</td>
			<td>EEG active electrode (10 in number)(Figure 2b)</td>
		</tr>
		<tr>
			<td>Computer with PC monitor</td>
			<td>Dell</td>
			<td></td>
			<td>Data processing and visual feedback (Figure 2)</td>
		</tr>
		<tr>
			<td>Softwares, EMG electrodes, NMES electrodes, and cables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a human-machine-interface integrating low-cost sensors with a neuromuscular electrical stimulation system for post-stroke balance rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

Figure 4 shows the eye gaze features that were extracted offline for the quantification of an able-bodied performance during a smooth pursuit task. The following features were extracted as shown in Table 1:
Feature 1 = percentage deviation between target stimulus position and the centroid of participant&#39;s fixation points when the stimulus is changing position in the horizontal direction....

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A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

The overall goal of this procedure is to apply video game sensor technology towards a low cost human machine interface for balance rehabilitation. This is accomplished by first setting up mobile brain body imaging, or MoBI equipment, and software. The second step is to place MoBI sensors on the body to capture physiological data.Next, an eye tracker is used to evaluate post stroke visual pursuit eye movements. Eye tracking will show that gaze related indices can help in delineating post-stroke oculomotor deficits, which may be relevant for planning visuomotor balance therapy. Then, sensors for neuromuscular electrical stimulation, or NMES, assisted in visuomotor balance therapy, under MoBI, are calibrated.The final step is to collect multi-sensor data from low cost sensors during visuomotor balance therapy. We are extending this technology for our home base rehabilitation for stroke patients who suffer from balance loss. Our system includes gaze based, prognostic dual, and one gaze spectrum as an important ingredient in visuomotor balance therapy.So this paradigm shifting technology captures how the visual system, the motor system, and the balance systems walks together after stroke and tries to rehabilitate them through training. So in principle, this technic can be used in other conditions, like Autism Spectrum Disorder, Parkinsonism, Traumatic Brain Injury, and also psychomotor disturbance due to depression. All can be rehabilitated in this training system.Begin by obtaining a projection screen to display the visual bio feedback. Adjust the height so that the center of the screen will be at the subject's eye level. Then place the motion caption sensor in front of the projection screen and aim it at the volume of motion capture.Confirm that the volume of motion capture is 1.5 meters to 2.5 meters in front of the motion capture sensor. Next, place the balance board on the floor, about 2.0 meters away from the motion capture sensor. Leave enough room around the balance board to ensure full body movement.To begin sensor placement for electromyography, or EMG, in the NMES system, ask the subject to sit on a chair. Place the EMG and NMES electrodes bilaterally on the Medial Gastrocnemius and Tibialis Anterior muscles of the subject. Then, connect the electrodes to the wireless NMES simulator system.Next, place the electroencephalogram, or EEG cap, on the subject's head by following the international 10-20 system. Use conductive paste to adhere the electrodes, seen here to the scalp, before connecting them to the wireless EEG headset. Place two passive EEG electrodes above and below one eye to capture the vertical electrooculogram, or EOG.Add another two to the outer campus of each eye to capture the horizontal EOG. Finally, place two passive EEG electrodes on the subject's earlobes to serve as EEG reference electrodes. Begin by placing the eye tracker just below the visual feedback computer monitor and connect it to the visual feedback computer using a USB 3 port.Then, ask the subject to sit at the desk at a distance of 50 centimeters from the eye tracker, adjust the visual feedback computer monitor so that the subject's eyes are at the center of the monitor. In the visual feedback computer, open the eye tribe server and eye tribe when-you-eye to ensure whether the subject's eyes are detected by the Gooey and to perform the eye tracker calibration routine subsequently. Next, ask the subject to look straight at the visual feedback computer monitor for visual cues.Run the eye tracker calibration routine by clicking on the calibrate tab in the Gooey. Run the visual underscore stimulus dot exc in the smart eye folder to execute the virtual reality based interface. Subsequently, run the smart eye dot exc program present in the smart eye folder to acquire the subject's eye gaze data that is synchronized with the virtual reality based task.Ask the subject to follow the fixed and moving visual stimuli at various positions on the visual feedback computer screen in order to evaluate post stroke pursuit eye movements. First, connect the eye tracker and balance board sensors to the visual feedback computer. Turn on the balance board, or BB sensor.Press the button on the BB sensor to make the remote discoverable in the menu. Click on the show or hide icon in the system's task bar, and click bluetooth device icon. Then, click on the added device option and pair the BB as a bluetooth device without using the code to the visual feedback computer.Once the balance board is connected to the visual feedback computer, open the VBT folder, and run the We BB interface dot m file to establish Matt lab BB interface. Have the subject stand on the balance board. Click OK to begin the calibration, then enter the subject's weight and click OK again.Next, connect and turn on the motion capture sensor to the data acquisition computer and ensure that it has fully booted, which is indicated by the green light. Open the LSL folder and start Mocap software to begin streaming of the motion capture sensor data. Turn on the EEG and EOG data acquisition systems connected to the data acquisition computer.To do this, first open the LSL folder and open open vibe acquisition server with LSL dot cmd. Finally, select emotive EPOC from the menu for EEG and EOG and configure the module by clicking on driver properties. Then, click on connect and click on play to start the acquisition server.Begin by asking the subject to stand on the balance board and attach a safety harness. Set a minimum baseline NMES level for upright standing by setting the stimulation frequency at 20 hurtz. Then, increase the pulse width and current level in the NMES software installed in the data acquisition computer until upright standing has been achieved.For NMES assisted VBT, ask the subject to sit on a chair, facing the motion capture sensor with their feet on the balance board. Run the calib sensors program in the data collect folder in order to collect multi sensor calibration data. Finally, ask the subject to perform self initiated maximal reach movements in different directions that affect center of mass location on the visual feedback.Begin by running the collect baseline program in the data collect folder to collect baseline resting state, eyes open, multi sensor data. To do this, ask the subject to stand still for two minutes and look straight at the center of mass location on the visual feedback. Then, run the collected VBT program in the data collect folder to collect sensor data during VBT.Connect the visual feedback computers video output to the projection screen and run the smart IVRT tasks dot exe file in the VBT folder in the visual feedback computer to launch the smart IVRT tasks Gooey. Then, ask the subject to pay attention to the audio and visual prompt on the computer screen and steer the cursor as fast as possible towards randomly presented peripheral targets. Following the Move Phase, ask the subject to hold the cursor at the target location for one second.Following the peripheral hold phase, ask the subject to return back to the central hold position while the system acquires the subject's two d coordinates of center of pressure and the two d coordinates of the gaze data. The eye gaze features were collected with the eye tracker to quantify a subjects performance during a smooth pursuit task for later comparison with post stroke VBT data. Then, for VBT, the protocol used to modify functional reach task to quantify the subject's ability to volitionally shift their center of pressure position as quickly as possible without losing balance, while cued with visual feedback.Here, EOG data shows that during VBT, the FD ratio, or ratio of fixation duration on the target, and the fixation duration on the cursor, increased while the normalized mean squared error decreased. While attempting this procedure it is very important to consider the safety and the comfort of the stroke patient. We expect that a virtual reality based balance rehabilitation system will be potent to address balance disorders of stroke patients.So following this procedure, other additional clinical measures, like the clinical measure of balance and mobility can be far from to comprehensively know how quantitive measures compared to other standard clinical parameters so that we can get a comprehensive picture of functional deficit of a stroke patient and we can comprehensively address this issue in the rehabilitation.

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Research

JoVE Journal

Neuroscience

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy 1. OHCs are able to change shape &#x2014;elongate, shorten and bend&#x2014; in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals 2.
OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization 2, 3. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) 4. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins 5, 6. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research 7.
The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) 8. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches 9-11. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques 12. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. 
We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.

A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

Assessment of Neuromuscular Function Using Percutaneous Electrical Nerve Stimulation

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels). The present protocol describes how this method can be used to stimulate the posterior tibial nerve that activates plantar flexor muscles. Percutaneous electrical nerve stimulation consists of inducing an electrical stimulus to a motor nerve to evoke a muscular response. Direct (M-wave) and/or indirect (H-reflex) electrophysiological responses can be recorded at rest using surface electromyography. Mechanical (twitch torque) responses can be quantified with a force/torque ergometer. M-wave and twitch torque reflect neuromuscular transmission and excitation-contraction coupling, whereas H-reflex provides an index of spinal excitability. EMG activity and mechanical (superimposed twitch) responses can also be recorded during maximal voluntary contractions to evaluate voluntary activation level. Percutaneous nerve stimulation provides an assessment of neuromuscular function in humans, and is highly beneficial especially for studies evaluating neuromuscular plasticity following acute (fatigue) or chronic (training/detraining) exercise.

We present a protocol to assess changes in neuromuscular function. Percutaneous electrical nerve stimulation is a non-invasive method that evokes muscular responses. Electrophysiological and mechanical properties of these responses permit the evaluation of neuromuscular function from brain to muscle (supra-spinal, spinal and peripheral levels).

Percutaneous electrical nerve stimulation is a non-invasive method commonly used to evaluate neuromuscular function from brain to muscle (supra-spinal, ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ex vivo preparation of the levator auris longus (LAL) is used because it is a thin muscle that provides easy visualization of the neuromuscular junction for microelectrode impalement at the motor endplate. This method allows for the recording of spontaneous miniature endplate potentials and currents (mEPPs and mEPCs), nerve-evoked endplate potentials and currents (EPPs and EPCs), as well as the membrane properties of the motor endplate. Results obtained from this method include the quantal content (QC), number of vesicle release sites (n), probability of vesicle release (prel), synaptic facilitation and depression, as well as the muscle membrane time constant (&#964;m) and input resistance. Application of this technique to mouse models of human disease can highlight key pathologies in disease states and help identify novel treatment strategies. By fully voltage-clamping a single synapse, this method provides one of the most detailed analyses of synaptic transmission currently available.

Levator Auris Longus Preparation for Examination of Mammalian Neuromuscular Transmission Under Voltage Clamp Conditions

The protocol described in this paper uses the mouse levator auris longus (LAL) muscle to record spontaneous and nerve-evoked postsynaptic potentials (current-clamp) and currents (voltage-clamp) at the neuromuscular junction. Use of this technique can provide key insights into mechanisms of synaptic transmission under normal and disease conditions.

This protocol describes a technique to record synaptic transmission from the neuromuscular junction under current-clamp and voltage-clamp conditions. An ...