This study was funded by the Christian Doppler Research Foundation of the Austrian Council for Research and Technology Development and the Austrian Federal Ministry of Science, Research and Economy. We are grateful to Aron Cserveny for preparation of the illustrations included in the manuscript and to Frontiers in Neuroscience for permission of reproducing the data presented in the original article12.

<ol>
	<li>Bertelli, J. A., Ghizoni, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Results+and+current+approach+for+Brachial+Plexus+reconstruction.">Results and current approach for Brachial Plexus reconstruction.</a> Journal of Brachial Plexus and Peripheral Nerve Injury. 6 (1), 2 (2011).</li><li>Birch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+lesions+of+the+brachial+plexus.">Traction lesions of the brachial plexus.</a> British Journal of Hospital Medicine. 32 (3), 140-143 (1984).</li><li>Narakas, A. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+neurofeedback+and+electromyographic-biofeedback+therapy+on+improving+hand+function+in+stroke+patients.">Effect of neurofeedback and electromyographic-biofeedback therapy on improving hand function in stroke patients.</a> Topics in Stroke Rehabilitation. 21 (2), 137-151 (2014).</li><li>Pfeufer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Training+with+biofeedback+devices+improves+clinical+outcome+compared+to+usual+care+in+patients+with+unilateral+TKA:+a+systematic+review.">Training with biofeedback devices improves clinical outcome compared to usual care in patients with unilateral TKA: a systematic review.</a> Knee Surgery, Sports Traumatology, Arthroscopy. 27 (5), 1611-1620 (2018).</li><li>Huang, H., Lin, J. J., Guo, Y. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+muscle+reinnervation+for+real-time+myoelectric+control+of+multifunction+artificial+arms.">Targeted muscle reinnervation for real-time myoelectric control of multifunction artificial arms.</a> JAMA. 301 (6), 619-628 (2009).</li><li>Miller, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+a+six+degree+of+freedom+prosthetic+arm+after+targeted+muscle+reinnervation+surgery.">Control of a six degree of freedom prosthetic arm after targeted muscle reinnervation surgery.</a> Archives of Physical Medicine and Rehabilitation. 89 (11), 2057-2065 (2008).</li><li>Kuiken, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+reinnervation+for+enhanced+prosthetic+arm+function+in+a+woman+with+a+proximal+amputation:+a+case+study.">Targeted reinnervation for enhanced prosthetic arm function in a woman with a proximal amputation: a case study.</a> Lancet. 369 (9559), 371-380 (2007).</li><li>Scheme, E., Englehart, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromyogram+pattern+recognition+for+control+of+powered+upper-limb+prostheses:+state+of+the+art+and+challenges+for+clinical+use.">Electromyogram pattern recognition for control of powered upper-limb prostheses: state of the art and challenges for clinical use.</a> Journal of Rehabilitation Research &amp; Development. 48 (6), 643-659 (2011).</li><li>Simon, A. M., Lock, B., Stubblefield, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient+training+for+functional+use+of+pattern+recognition-controlled+prostheses.">Patient training for functional use of pattern recognition-controlled prostheses.</a> Journal of Prosthetics and Orthotics. 24 (2), 56-64 (2012).</li><li>Simon, A. M., Hargrove, L. J., Lock, B. A., Kuiken, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+Achievement+Control+Test:+evaluating+real-time+myoelectric+pattern-recognition+control+of+multifunctional+upper-limb+prostheses.">Target Achievement Control Test: evaluating real-time myoelectric pattern-recognition control of multifunctional upper-limb prostheses.</a> Journal of Rehabilitation Research &amp; Development. 48 (6), 619-627 (2011).</li><li>Johnson, S. S., Mansfield, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prosthetic+training:+upper+limb.">Prosthetic training: upper limb.</a> Physical Medicine and Rehabilitation Clinics of North America. 25 (1), 133-151 (2014).</li><li>Kapelner, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+Unit+Characteristics+after+Targeted+Muscle+Reinnervation.">Motor Unit Characteristics after Targeted Muscle Reinnervation.</a> PLoS ONE. 11 (2), 0149772 (2016).</li><li>Sturma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surface+EMG+test+tool+to+measure+proportional+prosthetic+control.">A surface EMG test tool to measure proportional prosthetic control.</a> Biomedizinische Technik. Biomedical Engineering. 60 (3), 207-213 (2015).</li><li>Prahm, C., Kayali, F., Sturma, A., Aszmann, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PlayBionic:+Game-Based+Interventions+to+Encourage+Patient+Engagement+and+Performance+in+Prosthetic+Motor+Rehabilitation.">PlayBionic: Game-Based Interventions to Encourage Patient Engagement and Performance in Prosthetic Motor Rehabilitation.</a> PM&amp;R. 10 (11), 1252-1260 (2018).</li><li>Roche, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Structured+Rehabilitation+Protocol+for+Improved+Multifunctional+Prosthetic+Control:+A+Case+Study.">A Structured Rehabilitation Protocol for Improved Multifunctional Prosthetic Control: A Case Study.</a> Journal of Visualized Experiments. (105), e52968 (2015).</li></ol>

The authors have nothing to disclose.

Biofeedback approaches have been widely used in the rehabilitation of several neuromuscular disorders, ranging from (hemi)-plegic conditions resulting from central pathologies such as brain hemorrhage and stroke18,19 to various musculoskeletal degeneration or injury and their surgical therapy20,21,22. Interestingly, the concept of structured biofeedback has not been implemented in clinical practice for peripheral nerve injuries. However, precisely in the rehabilitation of complex nerve injuries, practice, repetition, and structured training programs with appropriate biofeedback are necessary to establish correct motor patterns23.
Here, and in a previous study12, we presented a structured rehabilitation protocol using sEMG biofeedback for patients with lack of biological treatment alternatives eligible for prosthetic hand replacement, a concept today known as bionic reconstruction. The most apparent advantage of using a sEMG biofeedback set-up in the context of bionic reconstruction arises from the exact definition of sEMG hotspots, i.e., skin locations, where a relatively high amplitude of EMG activity can be measured transcutaneously. Various motor commands may be attempted alternately, as the sensors can easily be moved along the entire forearm, and - in case of missing detectable muscle function in the forearm - also in the upper arm and shoulder girdle. When a patient is asked to attempt to contract the muscles intended to perform a specific action (such as extending the wrist), an electrode can be placed, where (weak) muscle contraction is palpated by the examiner. Observing the EMG signal on the computer screen, one can easily determine whether the signal&#39;s amplitude consistently increases, when the patient attempts to contract this muscle. If the amplitude is not high enough or the signal is inconsistent, other motor commands with the same electrode position may be attempted. As oppose to needle EMG, this procedure is non-invasive, not painful and can be repeated for all muscles/muscle groups in the arm. Testing various motor commands at different muscle locations allows to identify the EMG hotspots, with the highest amplitude and reproducible activity associated with a specific motor action. After identification of the strongest EMG signals, these may be trained using sEMG biofeedback with regards to signal separation (co-activation of two or more EMG signals must not occur on the computer screen), signal strength (reflected by the EMG signal&#8217;s amplitude on the computer screen) and signal reproducibility (each attempt to contract the muscle must lead to an excursion of the respective EMG signal). At a later stage of training, EMG activity is directly translated into prosthetic function, first using a table top prosthesis (see Figure 3), which gives additional feedback to the patient allowing fine-tuning of grip strength, and then wearing the physical prosthesis.
In conventional amputees, a vast amount of literature has shown that targeted-muscle-reinnervation (TMR), i.e., the surgical transfer of residual arm nerves to alternative muscle sites in the chest and upper-arm, improves prosthetic function, since these re-innervated muscles serve as biological amplifiers of intuitive motor commands and provide physiologically appropriate EMG signals for prosthetic hand, wrist and elbow control24,25,26,27. Using pattern-recognition control systems, EMG data extracted from numerous sEMG signals placed over the skin of these re-innervated muscles can be decoded and translated to specific, reproducible motor outputs, which provides more reliable myoelectric prosthesis control28,29,30. Because the number of EMG signal sites and the myoelectric activity of the muscles in patients with brachial plexus avulsion injury are very limited, pattern recognition algorithms may not be used as is done for conventional amputees8. Still, with further research and improved technology, these systems may be able to extract more information on the existing faint muscle signals and therefore improve prosthetic function also in this peculiar patient group.
While the presented protocol is considered a guideline, details need to be adapted depending on the patient and the available equipment. Due to aberrant re-innervation occurring after such nerve injuries, motor commands do not necessarily result in the activation of anatomically &#8220;correct&#8221; muscles12. For example, the authors observed EMG activity at the forearm flexor compartment, while patients were attempting to open their hand. Therefore, various motor commands should be tested in order to identify EMG signals. Additionally, the residual muscular function (although in all cases too weak to generate useful hand movements) might largely vary across patients and cause variations in the required training time as shown in Table 2. Further, the choice of the prosthetic device and the number of electrodes used for control change the requirements for the precision of signal separation, the signal amplitude and the need of co-contraction. All of this needs to be taken into account during signal training, hybrid prosthesis training and actual prosthetic training, as it is also recommended in standard prosthetic training of amputees31. Regarding the devices used for sEMG biofeedback training, the authors consider devices suitable if they can simultaneously display the number of signals needed for prosthetic control, give real-time feedback, and can be either connected to a computer or display the signals on a screen themselves. Devices that allow adjusting the signal gain during training are preferred.
After rehabilitation, all patients were able to use their prosthesis during daily life activities and were satisfied with the decision to have their functionless hand replaced with a prosthetic device12. This functional improvement was reflected by significant increases in the mean ARAT scores from 2.83 &#177; 4.07 to 25.00 &#177; 10.94 (p = 0.028).
From our perspective, sEMG biofeedback set-ups present valuable tools to facilitate the cognitively demanding process of motor recovery associated with nerve injury and bionic reconstruction. The identification of optimal EMG electrode positioning and the testing of various motor commands with direct visualization of muscle activity is greatly simplified using sEMG biofeedback in a clinical set-up. Although sEMG biofeedback may also be used in the rehabilitation of biological upper limb function10,12, its application in the process of bionic reconstruction is considered particularly effective. Most importantly, the sEMG signals activated during training later reflect the electrode positions within the prosthetic socket, which is individually customized for each patient. Therefore, repetitive activation of these signals during training most likely increases future prosthetic handling and manual capacity. Direct visualization of this muscle activity also allows a patient to comprehend the concept of myoelectric hand control and he/she may follow the training progress more consciously.
In the future, our presented rehabilitation protocol might be extended with more advanced tools to enhance functional outcomes. This might include high density sEMG recordings to facilitate the process of electrode placement via activation heat maps32, further virtual solutions to evaluate EMG activity30,33, and serious games to enhance training motivation34. Additionally, novel technologies for prosthetic control, such as pattern recognition algorithms might also be used28,30,35. However, due to the reduced neuro-muscular interface, it is not clear whether currently commercially available systems designed for otherwise healthy amputees would significantly improve prosthetic function in this specific patient group. Future studies should evaluate the applicability and benefits of the listed novel technologies for the rehabilitation of patients with severe brachial plexus injuries. Additionally, controlled trials with higher patient numbers will also allow to demonstrate the positive effects of the current protocol using sEMG biofeedback with a higher level of evidence.

Global brachial plexus injuries including the traumatic avulsion of nerve roots from the spinal cord represent one of the most severe nerve injuries in humans and usually affect young, otherwise healthy patients in the prime of life1,2. Depending on the number of nerve roots avulsed, complete upper limb paralysis may ensue since the nerval connection from the brain to the arm and hand is disrupted. Traditionally, the avulsion of nerve roots has been associated with poor outcomes3. With microsurgical nerve techniques gaining ground within the last decades, surgical results have been improved and useful motor function in the shoulder and elbow are usually restored4,5. The intrinsic musculature in the hand, which lies most distally, typically undergoes fatty degeneration resulting in irreversible atrophy before regenerating axons may reach it6. For such cases bionic reconstruction, which includes the elective amputation of the functionless &#34;plexus&#34;&#160;hand and its replacement with a mechatronic hand, has been described7,8. Residual muscle activity in a patient&#39;s forearm, which may be clinically insignificant (isometric contractions, M1 on the British Medical Research Council [BMRC] scale), is picked up from transcutaneous electrodes sensing electromyographic activity, which is then translated into various movements of a prosthetic hand9.
Enough surface electromyographic (sEMG) signals may be present upon initial consultation. In some cases, however, additional signals need to be established performing selective nerve and muscle transfers7. In either case, a structured rehabilitation protocol is needed to ensure sEMG signal consistency and subsequent optimal prosthetic function at the end of the process. A major challenge following nerve root avulsion and aberrant re-innervation as well as after nerve transfer surgery is the establishment of new motor patterns to allow volitional control over the target muscle. sEMG biofeedback methods have been widely used in the rehabilitation of stroke10. This method allows direct visualization of muscular activity that would otherwise be unnoticed due to muscular weakness and/or co-activation of antagonists. It thereby encourages patients to train their weak muscles, while providing precise feedback on the correct execution of motor tasks11.
In a recent publication we have shown for the first time that sEMG biofeedback may also be used in the rehabilitation of complex peripheral nerve injuries12. We believe that sEMG biofeedback is an extremely useful method to make a patient aware of the advancing re-innervation process after nerve transfer surgery. Also, faint muscle activity, which formerly was of no use to the patient, may be trained and strengthened for later prosthetic control using sEMG biofeedback, which allows concrete visualization of otherwise unnoticed muscle activity to both clinician and patient. The training progress may therefore be well comprehended and documented. Additionally, the use of direct feedback on muscle activity allows the clinician to correlate various motor commands with the associated signal amplitude and consistency, establishing the best motor strategies to allow robust prosthetic control in the future. In summary, the goal of this method is to facilitate the rehabilitation process by increasing a patient&#8217;s understanding, awareness and control of his/her sEMG signals, which will later drive a prosthetic hand.

<table>
	<tbody>
		<tr>
			<td>dry EMG electrodes</td>
			<td>Ottobock Healthcare, Duderstadt, Germany</td>
			<td>13E202 = 50</td>
			<td>The EMG electrodes used in this study were bipolar and included a ground. They can be used both for EMG training with the Myoboy and for the control of a prosthetic device.</td>
		</tr>
		<tr>
			<td>Myoboy</td>
			<td>Otto bock Healthcare, Duderstadt, Germany</td>
			<td>Myoboy</td>
			<td>This device that can be used as stand alone device or with a computer. It allows to display EMG activity while using the dry EMG electrodes that can also be impeded in the prosthetic socket.</td>
		</tr>
		<tr>
			<td>SensorHand Speed</td>
			<td>Ottobock Healthcare, Duderstadt, Germany</td>
			<td></td>
			<td>All patients used this commercially available myoelectrical prosthesis as their standard prosthetic device and during functional testing. Fitting of patients undergoing this procedure is, however, not restricted to this device.</td>
		</tr>
		<tr>
			<td>Standard laptop with Microsoft operating system</td>
			<td></td>
			<td></td>
			<td>Usually, devices for EMG biofeedback connected to a computer do not require much computing power and thus work on any regular laptop</td>
		</tr>
		<tr>
			<td>TeleMyo 2400T G2</td>
			<td>Noraxon, US</td>
			<td></td>
			<td>A surface EMG biofeedback set-up used in our protocol, connected to TeleMyo-Software, which displays the recorded EMG activity as color-coded graphs on the computer screen</td>
		</tr>
		<tr>
			<td>wet EMG electrodes</td>
			<td>Ambu</td>
			<td>Ambu Blue Sensor VL Adhesive Electrodes</td>
			<td>These adhesive electrodes can be used in combination with many different EMG biofeedback devices, including the TeleMyo 2400T. While they cannot be moved easily, the wet contacts usually allow to detect very faint EMG signals as well.</td>
		</tr>
	</tbody>
</table>

surface electromyographic biofeedback as a rehabilitation tool for patients with global brachial plexus injury receiving bionic reconstruction

In patients with global brachial plexus injury and lack of biological treatment alternatives, bionic reconstruction, including the elective amputation of the functionless hand and its replacement with a prosthesis, has recently been described. Optimal prosthetic function depends on a structured rehabilitation protocol, as residual muscle activity in a patient&#8217;s arm is later translated into prosthetic function. Surface electromyographic (sEMG) biofeedback has been used during rehabilitation after stroke, but has so far not been used in patients with complex peripheral nerve injuries. Here, we present our rehabilitation protocol implemented in patients with global brachial plexus injuries suitable for bionic reconstruction, starting from identification of sEMG signals to final prosthetic training. This structured rehabilitation program facilitates motor relearning, which may be a cognitively debilitating process after complex nerve root avulsion injuries, aberrant re-innervation and extra-anatomical reconstruction (as is the case with nerve transfer surgery). The rehabilitation protocol using sEMG biofeedback aids in the establishment of new motor patterns as patients are being made aware of the advancing re-innervation process of target muscles. Additionally, faint signals may also be trained and improved using sEMG biofeedback, rendering a clinically &#34;useless&#34;&#160;muscle (exhibiting muscle strength M1 on the British Medical Research Council [BMRC] scale) eligible for dexterous prosthetic hand control. Furthermore, functional outcome scores after successful bionic reconstruction are presented in this article.

In six patients with severe brachial plexus injuries including multiple nerve root avulsions the presented rehabilitation protocol using sEMG biofeedback was successfully implemented. Detailed patient characteristics can be found in Table 1. Figure 2 demonstrates the various phases of the structured rehabilitation protocol and detailed explanations on its implementation.
To demonstrate improvements in hand function before and after bionic reconstruction, a standardized assessment evaluating global upper extremity function was performed at two time points: before elective amputation of the functionless &#34;plexus&#34;&#160;hand as well as after successful prosthetic reconstruction and rehabilitation. The Action Research Arm test (ARAT) was originally developed to assess global upper extremity motor function in patients with cognitive impairment of hand control16. The standardized approach of Yozbatiran et al.17 was used in our studies. The ARAT consists of four different sections, which include tasks close to daily living. The test is timed by the observer who also rates the task performance from 0&#8722;3, with 3 indicating normal function. A maximum of 57 points is attainable indicating unimpaired motor function16. The number of therapy sessions with sEMG biofeedback and detailed results for each patient can be found in Table 2.
Although patient satisfaction with the offered rehabilitation protocol using sEMG biofeedback was not directly measured, all six patients reported to find it extremely helpful in comprehending the re-innervation process following nerve transfer surgery and to train the contraction of muscles with very faint activity that formerly was of no clinical use to them.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Case number</td>
			<td>Sex, age (years)</td>
			<td>Type of accident</td>
			<td>Type of lesion</td>
			<td colspan="2">Surgeries to improve biotechnological interface after initial reconstructions have failed to improve hand function</td>
		</tr>
		<tr>
			<td>1</td>
			<td>m, 32</td>
			<td>Fall from height</td>
			<td>Avulsion of C7&#8722;T1; traction injury of the infraclavicular plexus</td>
			<td colspan="2">Elective amputation of the forearm</td>
		</tr>
		<tr>
			<td>2</td>
			<td>m, 32</td>
			<td>Motorcycle accident</td>
			<td>Rupture of all 3 trunci of the BP</td>
			<td colspan="2">Free gracilis muscle transferred to forearm extensor compartment &#38; neurotization of deep branch of radial nerve to obturator nerve; elective amputation of the forearm</td>
		</tr>
		<tr>
			<td>3</td>
			<td>m, 55</td>
			<td>Motorcycle accident</td>
			<td>Avulsion of C5&#8722;T1</td>
			<td colspan="2">Elective amputation of the upper arm</td>
		</tr>
		<tr>
			<td>4</td>
			<td>m, 38</td>
			<td>Motorcycle accident</td>
			<td>Extensive damage to roots&#160;C5&#8722;C8; avulsion of T1</td>
			<td colspan="2">Elective amputation of the forearm</td>
		</tr>
		<tr>
			<td>5</td>
			<td>m, 27</td>
			<td>Motorcycle accident</td>
			<td>Avulsion C8&#8722;T1</td>
			<td colspan="2">Elective amputation of the forearm</td>
		</tr>
		<tr>
			<td>6</td>
			<td>m, 43</td>
			<td>Motorcycle accident</td>
			<td>Avulsion of C6&#8722;T1</td>
			<td colspan="2">Transfer of triceps muscle to infraspinatous fossa and transfer of biceps muscle to supraclavicular fossa to improve prosthetic fitting; Elective amputation of the arm (shoulder exarticulation)</td>
		</tr>
	</tbody>
</table>
Table 1: Patient characteristics.&#160;In all patients, bionic reconstruction was initiated due to infeasibility of biological treatment alternatives. Surgeries to establish EMG signals in the fore- and upper arm may include selective nerve and muscle transfers, which will then drive a myoelectric prosthetic hand. Elective amputation is either performed at a transradial or transhumeral level, depending on the residual muscle activity. All selective nerve transfers performed in this patient group were successful. This table has been modified from Sturma et al.12&#160;and reproduced with permission from Frontiers in Neuroscience.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Case number</td>
			<td>ARAT at baseline</td>
			<td>ARAT at follow-up</td>
			<td colspan="2">Start of sEMG training</td>
			<td>Number of therapy sessions in total (30 min each)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>7</td>
			<td>35</td>
			<td colspan="2">Immediately after first consultation</td>
			<td>24</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0</td>
			<td>15</td>
			<td colspan="2">Training with one signal immediately after first consultation; second signal was available 9 months after free gracillis transfer + nerve transfer</td>
			<td>30</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0</td>
			<td>19</td>
			<td colspan="2">Immediately after first consultation</td>
			<td>16</td>
		</tr>
		<tr>
			<td>4</td>
			<td>1</td>
			<td>22</td>
			<td colspan="2">Immediately after first consultation</td>
			<td>20</td>
		</tr>
		<tr>
			<td>5</td>
			<td>9</td>
			<td>42</td>
			<td colspan="2">Immediately after decision to aim for a bionic reconstruction as biologic reconstruction failed</td>
			<td>20</td>
		</tr>
		<tr>
			<td>6</td>
			<td>0</td>
			<td>17</td>
			<td colspan="2">Immediately after first consultation</td>
			<td>22</td>
		</tr>
		<tr>
			<td>Mean (&#177; SD)</td>
			<td>2.83 &#177; 4.07</td>
			<td>25.00 &#177; 10.94</td>
			<td colspan="2"></td>
			<td>22 &#177; 4.32</td>
		</tr>
	</tbody>
</table>
Table 2:&#160;ARAT scores and number of therapy sessions.&#160;In the Action Research Arm test (ARAT), patients initially showed negligible upper limb function (mean 2.83, of a maximum of 57 points attainable). Useful function was restored after bionic reconstruction (mean 25.00, of 57). This table has been modified from Sturma et al.12&#160;and reproduced with permission from Frontiers in Neuroscience.

Watch this Scientific Journal Video about Surface Electromyographic Biofeedback as a Rehabilitation Tool for Patients with Global Brachial Plexus Injury Receiving Bionic Reconstruction at JoVE.com

Surface Electromyographic Biofeedback as a Rehabilitation Tool for Patients with Global Brachial Plexus Injury Receiving Bionic Reconstruction

Optimal functional outcomes after bionic reconstruction in patients with global brachial plexus injury depend on a structured rehabilitation protocol. Surface electromyographic guided training may improve the amplitude, separation and consistency of EMG signals, which - after elective amputation of a functionless hand - control and drive a prosthetic hand.

In patients with global brachial plexus injury and lack of biological treatment alternatives, bionic reconstruction, including the elective amputation of ...

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11.3K Views.  Medical University of Vienna. Optimal functional outcomes after bionic reconstruction in patients with global brachial plexus injury depend on a structured rehabilitation protocol. Surface electromyographic guided training may improve the amplitude, separation and consistency of EMG signals, which - after elective amputation of a functionless hand - control and drive a prosthetic hand.

Video: Surface Electromyographic Biofeedback as a Rehabilitation Tool for Patients with Global Brachial Plexus Injury Receiving Bionic Reconstruction

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 ...

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

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.

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 ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

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 ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask gait analysis consisted of a single walking task (Task 0), a simple motor dual-task (water-holding, Task 1), and a complex motor dual-task (crossing obstacles, Task 2). The task of crossing obstacles was considered to be equivalent to the combination of a simple walking task and a complex motor task as it involved more nervous system, skeletal movement, and cognitive resources. To eliminate heterogeneity in the results of the gait analysis of the stroke patients, the dual-task gait cost values were calculated for various kinematic parameters. The major differences were observed in the proximal joint angles, especially in the angles of the trunk, pelvis, and hip joints, which were significantly larger in the dual motor tasks than in the single walking task. This research protocol aims to provide a basis for the clinical diagnosis of gait function and an in-depth study of motor control in stroke patients with motor control deficits through the analyses of dual-motor walking tasks.

This paper presents a protocol specifically for dual motor task gait analysis in stroke patients with motor control deficits.

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask ...