This protocol was developed for research supported by the Natural Sciences and Engineering Research Council of Canada (grant RGPIN 401963).

The following protocol was approved by the Research Ethics Board at Holland Bloorview Kids Rehabilitation Hospital.
1. Motor Calibration
<ol>
	<li>Connect the microcontroller board to the computer using an USB port.</li>
	<li>Using the original microcontroller software, upload the custom script, &#34;Motor_and_AccelerometerTest.ino&#34; to the board using the USB connection by clicking the &#34;Upload&#34; icon, denoted by the circled right arrow.
	<ol>
		<li>Ensure that the vibration level is set to zer...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vibrotactile+stimulation+of+the+upper+leg:+Effects+of+location,+stimulation+method+and+habituation.">Vibrotactile stimulation of the upper leg: Effects of location, stimulation method and habituation.</a> Proc. Annu. Int. Conf. IEEE Eng. Med. Biol. Soc. , 1668-1671 (2011).</li><li>Rusaw, D., Hagberg, K., Nolan, L., Ramstrand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+vibratory+feedback+be+used+to+improve+postural+stability+in+persons+with+transtibial+limb+loss?.">Can vibratory feedback be used to improve postural stability in persons with transtibial limb loss?.</a> J. Rehabil. Res. Dev. 49 (8), 1239-1254 (2012).</li><li>Goodworth, A. D., Wall, C., Peterka, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+feedback+parameters+on+performance+of+a+vibrotactile+balance+prosthesis.">Influence of feedback parameters on performance of a vibrotactile balance prosthesis.</a> IEEE Trans. Neural Syst. Rehabil. Eng. 17 (4), 397-408 (2009).</li><li>Asseman, F., Bronstein, A. M., Gresty, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+vibrotactile+feedback+of+instability+to+trigger+a+forward+compensatory+stepping+response.">Using vibrotactile feedback of instability to trigger a forward compensatory stepping response.</a> J. Neurol. 254 (11), 1555-1561 (2007).</li><li>Fan, R. E., Culjat, M. 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A., Verrillo, R. T., Checkosky, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+channels+mediate+the+mechanical+aspects+of+touch.">Four channels mediate the mechanical aspects of touch.</a> J. Acoust. Soc. Am. 84 (5), 1680-1694 (1988).</li><li>Giggins, O. M., Persson, U. M., Caulfield, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofeedback+in+rehabilitation.">Biofeedback in rehabilitation.</a> J. Neuroeng. Rehabil. 10 (1), 60 (2013).</li><li>Shull, P. B., Jirattigalachote, W., Hunt, M. A., Cutkosky, M. R., Delp, S. 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The purpose of this protocol is to provide the framework for evaluating stimulation parameters in vibrotactile ASF applications. Specifically, it examines the effects of vibration frequency, amplitude, location, and sequence on user sensorimotor response. This framework can be built upon and expanded to incorporate additional or alternative types of user response that may be more clinically relevant, such as bending a joint or shifting weight from one leg to another. These types of changes would require slightly differen...

Artificial sensory feedback (ASF) can be defined as the practice of providing real-time biological information to individuals, often compensating for compromised proprioception or other sensory mechanism. ASF has been long used in the realm of rehabilitation of injured or disabled persons to assist in recovering of aspects of physical function and movement1-3, allowing individuals to control physical processes that once were an involuntary response of the autonomic nervous system4. A subcategory of ASF, biomechanical biofeedback, uses external sensors to measure parameters relating to balance or gait kinematics, and communicate this information to the individual through some sort of applied stimulus. An increasingly popular approach to biomechanical feedback employs small vibrating motors, or tactors, placed at different parts of the body to provide spatial as well as temporal feedback. Previous literature has showed promising results supporting the use of vibrotactile feedback in applications to individuals with lower-limb amputations, vestibular impairments, and aging-related loss of balance5-9.
A thorough understanding of the mechanisms controlling an individual&#39;s perception and response to specific stimuli is necessary for informing effective implementation of ASF systems for different applications. For vibrotactile feedback, chief among these mechanisms are proprioception and the sensorimotor response, specifically the user sensitivity to the applied vibrations and the time required to execute the desired reaction. Any sensory information communicated through vibration stimuli must be encoded as specific combinations of vibration frequency, amplitude, location, and sequence. Therefore, design of vibrotactile ASF systems should select combinations of parameters to maximize user&#39;s perception and interpretation of the stimuli, as well as the timeliness and accuracy of the resulting motor response. The goal of this protocol is to provide a platform from which to evaluate response times and response accuracy to various vibrational stimuli to inform the design of ASF systems for use with different sensory-impaired populations.
The methods described here builds on prior research exploring human perception of tactile and vibrotactile feedback3,5,6, and was developed for use in two previous studies10,11. The latter two studies employed this protocol to examine the effects of vibration frequency and location on the accuracy and timeliness of user responses in lower-limb amputees, showing that both parameters significantly affect the outcome measures, and that a high degree of response accuracy can be achieved. These results can be used to inform the ideal placement of tactors in future studies and clinical applications of vibrotactile ASF systems. Other recent work by Crea et al.12 examined user sensitivity to changes in vibration patterns applied to the thigh during walking, using verbal responses to signify perceived changes to the vibration patterns, rather than a motor response. While these verbal responses can be used to measure detection accuracy, they do not account for errors and delays that may be present in the motor control process.
The primary setup for the following experiments consists of a number of vibrating motors connected to pulse-width-modulated output pins of a microcontroller board. The board is, in turn, controlled through a Universal Serial Bus (USB) connection to a computer running commercially available system design software. The motors require an additional amplifying circuit to ensure sufficient voltage and current is supplied over a wide range of vibration frequencies. An example amplifier circuit is shown in Figure 1. The bipolar junction transistor (BJR) in the figure can be replaced with smaller metal-oxide-semiconductor field-effect transistor (MOSFET) for more efficient operation and smaller size. Similarly, the entire amplifying circuit can be replaced by an off-the-shelf haptic motor driver to provide additional control and reduced size. Each motor requires its own circuit, and using the equipment listed in this paper, up to ten motors can be controlled by a single microcontroller board.
<img alt="Figure 1" src="/files/ftp_upload/54223/54223fig1.jpg" /> 
Figure 1. Motor Wiring. (A) The amplification circuit for a single vibration motor is shown. Each motor requires a separate circuit and must be connected to a unique PWM output port on the microcontroller. The VDD here represents the 3.3 V power supplied by the microcontroller board, and the resistor R2 serves as a pull-down resister to ensure the transistor switch remains open when zero voltage is applied. (B) An example of the physical wiring of two motors. Although eight individual amplification circuits are shown, only two are connected to vibration motors. In this protocol R1 = 4.7 k&#937; and R2 = 100 k&#937;. <a href="https://www.jove.com/files/ftp_upload/54223/54223fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="849">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vibrating Pager Motors</td>
			<td style="width:107px;">Precision Microdrives</td>
			<td style="width:119px;">Model 310-101</td>
			<td style="width:407px;">Coin eccentric rotating mass motors.&#160; As many as necessary to test all locations and interactions of interest</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Tri-axis Accelerometer</td>
			<td style="width:107px;">Dimension Engineering</td>
			<td style="width:119px;">ADXL 335</td>
			<td style="width:407px;">Advanced analog accelerometer. 500Hz bandwidth, 3.5-15V input.&#160; Designed for motion, tilt, and slope measurement, as well as vibration and shock sensing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Arduino Uno</td>
			<td style="width:107px;">Arduino</td>
			<td style="width:119px;">DEV-11021</td>
			<td style="width:407px;">Microcontroller board for communicating with the tri-axis accelerometer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Arduion Mega 2560</td>
			<td style="width:107px;">Arduino</td>
			<td style="width:119px;">DEV-11061</td>
			<td style="width:407px;">Microcontroller board for interfacing with the vibration motors.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LabVIEW</td>
			<td style="width:107px;">National Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:407px;">Data acquisition software used to control motors and display accelerometer signals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Arduino IDE Software</td>
			<td style="width:107px;">Arduino</td>
			<td style="width:119px;">v. 1.6.5</td>
			<td style="width:407px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Push-Button</td>
			<td style="width:107px;">Bridges</td>
			<td style="width:119px;">Buddy Button</td>
			<td style="width:407px;">Wired switch featuring a 2.5in/6.35cm activation surface that provides an auditory click and tactile feedback.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optional:</td>
			<td style="width:107px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dedicated haptic motor driver</td>
			<td style="width:107px;">Texas Instruments</td>
			<td>DRV2605L</td>
			<td style="width:407px;">Can be used to replace the entire amplification circuit described in Step 1.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Flexible wearable goniometer</td>
			<td style="width:107px;">Biometrics Ltd.</td>
			<td style="width:119px;">SG110</td>
			<td style="width:407px;">Twin axis flexible goniometers to measure angles in up to two planes of movement that can be used in lieu of the push button to measure joint movement in response to stimuli. 
			www.biometricsltd.com/gonio.htm</td>
		</tr>
	</tbody>
</table>

a method for evaluating timeliness and accuracy of volitional motor responses to vibrotactile stimuli

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments. Effective design of these ASF systems requires an in-depth understanding of how the parameters of specific feedback mechanism affect user perception and reaction to stimuli. This article presents a method for applying vibrotactile stimuli to human participants and measuring their response. Rotating mass vibratory motors are placed at pre-defined locations on the participant's thigh, and controlled through custom hardware and software. The speed and accuracy of participants' volitional responses to vibrotactile stimuli are measured for researcher-specified combinations of motor placement and vibration frequency. While the protocol described here uses push-buttons to collect a simple binary response to the vibrotactile stimuli, the technique can be extended to other response mechanisms using inertial measurement units or pressure sensors to measure joint angle and weight bearing ratios, respectively. Similarly, the application of vibrotactile stimuli can be explored for body segments other than the thigh.

Figure 4 illustrates the calibration curves identifying the PWM value for a 180 Hz vibration frequency of a single motor. Starting at a 50% duty cycle, the PWM values are iterated until the primary frequency spike occurs at 180 Hz. Successful calibration trials should show a clear spike at the primary vibration frequency. Poor fixation of the accelerometer to the motor, or of the motor to a support surface may result in a more diffuse FFT without a clear spike. In this situation, the calibration trial sh...

Watch this Scientific Journal Video about A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli at JoVE.com

A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and frequency.

Artificial sensory feedback (ASF) systems can be used to compensate for lost proprioception in individuals with lower-limb impairments.  Effective design ...

The overall goal of this experimental protocol is to evaluate how quickly and accurately an individual can respond to specific types of vibrotactile stimuli. This method could help answer key questions in the rehabilitation engineering field such as what type of sensory information can be communicated through vibrotactile feedback and what is the best way to represent sensory information through vibrotactile signals. The main advantage of this technique is that it is very flexible for different bio feedback applications and can be easily adapted for use with a variety of bio sensors such as pressure sensors for measuring reaction force, or goniometers for measuring joint angles.Though this method provides insight into how people respond to vibrations apply to specific areas of the thigh, it can also be extended to other areas of the body. By testing different tactile configurations, we can better understand what types of information can be communicated through bio feedback. Demonstrating this procedure will be Sam Shi a graduate student in our lab.Begin the motor calibration by connecting the large microcontroller board to the computer using a USB port. Within the original microcontroller software, click the upload icon denoted by the circled right arrow to upload the custom script motor_test. ino to the board using the USB connection.Then connect the Z axis output of the triaxis accelerometer to one of the analog input ports of the smaller microcontroller board. Connect the positive and ground leads from the accelerometer to the thigh bolt and ground ports of the microcontroller board respectively. Next mount the accelerometer to the vibrating motor ensuring that its z axis is orthogonal to the flat surface of the motor and then place the motor on a hard surface.Open the motor_calibration. vi file in the data question software and connect the smaller microcontroller for the accelerometer to the computer through a USB port. Then using the fields provided, specify the serial port for the microcontroller input as well as the sampling rate of 500 hertz and 1000 samples to collect.Using the fast 48 transform display on the motor_calibration. vi interface identify the largest peak and record the corresponding vibration frequency value. Repeat all of the steps thus far as well as manually record the pulse with modulated or PWM frequency relationship for each motor.Next open the experiment1. vi file for each motor. Right click on the dropdown frequency menu and select property.Under the edit items tab, use the table to enter the desired frequencies and corresponding PWM levels. Select okay to exit. Finally, after all motors have been calibrated use double sided tape to mount each one to the thigh.Specifically place one motor on each of the anterior, posterior, medial, and lateral surfaces of the thigh approximately midway between the greater trochanter and lateral femoral condyle. To begin the first experiment, connect the push button directly to one of the computer's USB ports using a serial to USB connector and ensure that all required drivers are installed. Open the experiment1.vi interface and select the file to record the results. Next, use the dropdown menus in the software interface to select the push button input ports and motor frequencies. Start the program.When the participant feels a vibration, have them press the push button with the leg where the feedback is applied. After the button has been pressed, confirm the response in the data acquisition software interface by noting that the clock has stopped counting. Finally select the new set of frequencies from the dropdown menus to reset the motors for the next trial.Begin the second experiment by connecting a second push button to another USB port using a serial USB connector. Then open the experiment2. vi data acquisition interface.In order to examine frequency sensitivity at different portions of the leg place a single motor on the thigh midway between the greater trochanter and the lateral femoral condyle on each of the anterior, posterior, lateral, and medial surfaces. Next click on the motor icons in the software interface to select specific motors to be activated as well as the desired sequence of frequencies. To test user sensitivity, to frequencies at 140 hertz, 180 hertz and 220 hertz start by selecting a single combination.For example 180 hertz followed by 140 hertz. Input the wait time and motor time. Then start the program.To test other frequency combinations, select a new test of frequencies such as 220 hertz, followed by 180 hertz and rerun the program. Finally have the participant press one of the two push buttons to choose whether the second perceived frequency was higher or lower than the first and observe the responses that are automatically recorded by the program. The results of the fast 48 transform of the acceleration data are shown here for a single motor undergoing calibration.Four trails were conducted to identify the PWM level corresponding to the 180 hertz vibration. When comparing able bodied individuals with three individuals with transfemoral amputations reaction times for the able bodied individuals significantly decreased as frequency went from 140 hertz to 220 hertz. While those for the individuals with amputations did not.Once mastered, this protocol can be completed in 30 minutes to an hour depending on the number of frequency location combinations tested. While attempting this procedure, it is important to ensure that the trackers maintain good contact with the thigh throughout the experiments. Care should be taken to confirm that the trackers are secure before each trial.While the protocol described here uses push buttons to record the user response, the method can be extended to other response mechanisms that are more physiologically relevant such as bending the knee. After its development, this method paved the way for researchers in the field or rehabilitation engineering to explore different strategies for communicating sensory information from a variety of sources through vibrotactile feedback in able bodied individuals as well as those with lower limb amputations. After watching this video, you should have a good understanding of how to evaluate the speed and accuracy of user responses to different combinations of vibrotactile stimulation frequency and location.

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7.2K ビュー.  Holland Bloorview Kids Rehabilitation Hospital. The overall goal of this experimental protocol is to evaluate how quickly and accurately an individual can respond to specific types of vibrotactile stimuli. This method could help answer key questions in the rehabilitation engineering field such as what type of sensory information can be communicated through vibrotactile feedback and what is the best way to represent sensory information through vibrotactile signals. The main advantage of this technique is that it is very flexible for differe...

ビデオ: A Method for Evaluating Timeliness and Accuracy of Volitional Motor Responses to Vibrotactile Stimuli

This article describes a technique for applying vibrotactile stimuli to the thigh of a human participant, and measuring the accuracy and reaction time of the participant&#39;s volitional response for various combinations of stimulation location and...

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Author Spotlight: Advancing Spinal Cord Stimulation - Exploring the Cellular Responses of Motor Neurons Through Patch-Clamp Electrophysiology 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability...

Understanding Motor Neuron Responses to Spinal Cord Stimulation

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evaluating targeting accuracy in the focal plane for an ultrasound-guided high-intensity focused ultrasound phased-array system

Evaluating Targeting Accuracy in the Focal Plane for an Ultrasound-guided High-intensity Focused Ultrasound Phased-array System

This study describes a protocol to evaluate the targeting accuracy in the focal plane of an ultrasound-guided high-intensity focused ultrasound phased-array...

designing microfluidic devices for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress...

pyokr: a semi-automated method for quantifying optokinetic reflex tracking ability

Author Spotlight: A Streamlined and Accessible Analysis Method to Quantify Optokinetic Reflex Tracking Responses

We describe here PyOKR, a semi-automated quantitative analysis method that directly measures eye movements resulting from visual responses to two-dimensional image motion. A Python-based user interface and analysis algorithm allows for higher throughput and more accurate quantitative measurements of eye-tracking parameters than previous methods.

Python-Based Tool for Quantitative Optokinetic Reflex Assessment in Animals

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patch-clamp recording to study the electrical responses of motor neurons to spinal cord stimulation

Patch-Clamp Recording to Study the Electrical Responses of Motor Neurons to Spinal Cord Stimulation  

four-way olfactometer assay: a method to assess odorant-cued responses in drosophila

Fruit flies have a complex olfactory system, can discriminate hundreds of odorants, and use their sense of smell to direct behavioral decisions. This video describes the four-way olfactometer assay, a method to study olfactory behavioral responses in flies. The featured protocol clip shows how to handle test flies and what are critical steps while performing the...

Four-Way Olfactometer Assay: A Method to Assess Odorant-Cued Responses in Drosophila

Haptic/Graphic Rehabilitation: Integrating a Robot into a Virtual Environment Library and Applying it to Stroke Therapy

Recent research that tests interactive devices for prolonged therapy practice has revealed new prospects for robotics combined with graphical and other forms of biofeedback. Previous human-robot interactive systems have required different software commands to be implemented for each robot leading to unnecessary developmental overhead time each time a new system becomes available. For example, when a haptic/graphic virtual reality environment has been coded for one specific robot to provide haptic feedback, that specific robot would not be able to be traded for another robot without recoding the program. However, recent efforts in the open source community have proposed a wrapper class approach that can elicit nearly identical responses regardless of the robot used. The result can lead researchers across the globe to perform similar experiments using shared code. Therefore modular "switching out"of one robot for another would not affect development time. In this paper, we outline the successful creation and implementation of a wrapper class for one robot into the open-source H3DAPI, which integrates the software commands most commonly used by all robots.

Recently, a vast amount of prospects have come available for human-robot interactive systems. In this paper we outline the integration of a new robotic device with open source software that can rapidly make possible a library of interactive functionality. We then outline a clinical application for a neurorehabilitation application.

Recent research that tests interactive devices for prolonged therapy practice has revealed new prospects for robotics combined with graphical and other ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...

Evaluating Primary Blast Effects In Vitro

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such blast-induced injuries is of great importance considering the recent trend towards the use of explosives in modern warfare and terrorist-related incidents. To fully understand blast-induced injury, we must first be able to replicate such blast events in a controlled environment using a reproducible method. In this technique using shock tube equipment, shock waves at a range of pressures can be propagated over live cells grown in 2D, and markers of cell viability can be immediately analyzed using a redox indicator assay and the fluorescent imaging of live and dead cells. This method demonstrated that increasing the peak blast overpressure to 127 kPa can stimulate a significant drop in cell viability when compared to untreated controls. Test samples are not limited to adherent cells, but can include cell suspensions,&#160;whole-body and tissue samples, through minor modifications to the shock tube setup. Replicating the exact conditions that tissues and cells experience when exposed to a genuine blast event is difficult. Techniques such as the one presented in this article can help to define damage thresholds and identify the transcriptional and epigenetic changes within cells that arise from shock wave exposure.

Evaluating Primary Blast Effects In Vitro

Understanding how cells are modulated by exposure to shock waves can help identify the mechanisms behind injuries triggered from blast events. This protocol uses custom-built shock tube equipment to apply shock waves at a range of pressures to cell monolayers and to identify the subsequent effects on cell viability.

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...