The authors thank Professor Winston Byblow and Harry Jordan for their valuable contribution to this work. This work was funded by the Health Research Council of New Zealand.

All research conducted with human participants must have human ethics approval by the appropriate institutional ethics committee and the study must be conducted in accordance with the declaration of Helsinki.
1. Patient Screening
<ol>
	<li>Screen all patients for PREP2 suitability within 72 h of stroke onset. 
	NOTE: Patients are suitable if they have had a unilateral ischemic or hemorrhagic stroke within the last 72 h, have new UL weakness, and are 18 years or older.</li>
</ol>
2. SAFE Score
NOTE: Ensure that patients with inattention or fatigue are focused on the arm to enable an accurate assessment of strength.
<ol>
	<li>Position the patient with their back fully supported and upright either in bed or in a chair and their paretic arm by their side with the elbow in extension.</li>
	<li>Demonstrate shoulder abduction. Ask the patient to lift their arm sideways and up towards their ear. Use the Medical Research Council (MRC) grades to score shoulder abduction strength. 
	NOTE: MRC grades are described as follows: 0 = no palpable muscle activity; 1 = palpable muscle activity but no movement; 2 = limited range of movement without gravity; 3 = full range of motion against gravity but no resistance; 4 = full range of motion against gravity and resistance but weaker than the other side; 5 = normal power.</li>
	<li>To score shoulder abduction grades 4 or 5, place your hand over the patient&#39;s arm, proximal to the elbow and apply resistance. 
	NOTE: The patient must be able to achieve full range of motion against resistance to be awarded a score of 4 or higher.</li>
	<li>Place the paretic forearm in pronation with the fingers fully flexed and provide support under the wrist.</li>
	<li>Demonstrate finger extension. Ask the patient to straighten their fingers and use the Medical Research Council grades to score finger extension strength.</li>
	<li>To score finger extension grades 4 or 5, apply resistance over the dorsum of the fingers, distal to the metacarpophalangeal joints, throughout the movement. 
	NOTE: The patient must be able to achieve full extension against resistance to be awarded a score of 4 or higher.</li>
	<li>To score fingers with unequal strength, use the majority rule. If three fingers have the same score, use this score. If two fingers have a lower score than the other two fingers, use the lower score.</li>
	<li>Add the MRC grades for shoulder abduction and finger extension together for a SAFE score out of 10. If the patient has a SAFE score of 5 or more on day 3 poststroke they can be assumed to have a functional corticospinal tract and TMS is not required. If the patient has a SAFE score less than 5 on day 3 poststroke, TMS is required to determine their MEP status.</li>
</ol>
3. Transcranial Magnetic Stimulation (TMS)
<ol>
	<li>Evaluate patient suitability for TMS.
	<ol>
		<li>Complete a TMS safety checklist with the patient to identify absolute and relative contraindications to TMS24. 
		NOTE: This information should be gathered through patient and family interview and from the medical records. See representative results section for more detail.</li>
		<li>Ask the patient&#39;s physician to review the TMS checklist and approve it if appropriate.</li>
		<li>On the day of the TMS test, review the patient&#39;s medical status with the clinical team and the patient to ensure there have been no changes since the checklist was signed. 
		NOTE: Events to consider include fall with head injury, seizure, if the patient has become medically unwell, is hypoglycemic, or has unstable blood pressure. Ensure the patient has taken all prescribed medication prior to the test.</li>
	</ol>
	</li>
	<li>Prepare the environment.
	<ol>
		<li>Remove furniture from around the bed. Move the bed away from the wall to allow space for the TMS unit.</li>
		<li>Place the TMS unit at the head of the bed towards the side opposite the paretic limb. Angle the TMS unit so that the person delivering TMS can easily see the screen.</li>
		<li>Test the TMS setup to check it is working appropriately. 
		NOTE: This protocol uses a single-pulse TMS unit. The electromyography (EMG) signal can be sampled at 2 kHz and filtered with 10 Hz high-pass and 1,000 Hz low-pass filters. The EMG equipment needs to be triggered by the TMS unit such that the EMG trace begins at least 50 ms prior to the TMS stimulus and ends at least 50 ms after the TMS stimulus.</li>
		<li>Ensure familiarity with the protocol for summoning emergency assistance in the TMS assessment room in case this is required.</li>
	</ol>
	</li>
	<li>Prepare the patient. 
	NOTE: Patients with intravenous (IV) lines, nasogastric feeding, or low concentrations of supplementary oxygen via a nasal cannula can be tested with TMS provided that they are considered medically stable by the treating physician. Supplemental oxygen should continue throughout the TMS session. Suspending and disconnecting nasogastric feeding and non-essential fluids through IV lines will make it easier to conduct the TMS assessment.
	<ol>
		<li>Remove any clothing covering the forearms. Remove any items covering the paretic wrist such as a watch or identification bracelet to enable the EMG electrode placement.</li>
		<li>Position the paretic arm on a pillow with the forearm pronated and fully supported from the elbow to the hand.</li>
		<li>Palpate the paretic forearm to locate the muscle belly for the extensor carpi radialis (ECR) muscle. Identify positions for two surface EMG electrodes over the muscle belly, allowing for factors such as the position of IV cannula or dressings. 
		NOTE: It is essential that at least one electrode is positioned over the muscle belly. This may require discussion with the nursing staff about repositioning dressings before the test if possible.</li>
		<li>Clean the skin at each electrode site with an alcohol skin-cleansing wipe. Shave each electrode site to remove any hair. Lightly abrade the electrode sites with an abrasive cream or tape. Take care with patients who have fragile skin and avoid any areas of broken skin.</li>
		<li>Securely apply self-adhesive disposable recording electrodes to each site.</li>
		<li>Locate the electrode sites for the first dorsal interosseus muscle (FDI). One electrode will be placed on the FDI muscle belly and one on the dorsum of the hand. 
		NOTE: Electrode placement may vary depending on patient factors such as the position of an IV cannula or dressings.</li>
		<li>Prepare the skin and apply the self-adhesive recording electrodes as previously described.</li>
		<li>Place the reference electrode strap around the arm just proximal to the elbow. Alternatively, prepare the skin and place a self-adhesive reference electrode over the lateral epicondyle of the humerus.</li>
	</ol>
	</li>
	<li>Position the patient in bed for the test.
	<ol>
		<li>Lower the bed rails. Move the patient as high up the bed as possible and towards the edge of the bed on the non-paretic side. 
		NOTE: The patient should only be moved by trained staff.</li>
		<li>Put the bed rail back up on the paretic side for safety. Remove the headboard of the bed if possible and remove any unused IV poles attached to the bed that may obstruct the coil position.</li>
		<li>Raise the head of the bed as high as possible. Position pillows behind the patient&#39;s back to bring them into an upright sitting position without their head contacting the bed. Do not put a pillow behind the head. If possible, raise the knees to prevent the patient from slipping down the bed during testing.</li>
		<li>Ensure the paretic forearm is in pronation and fully supported by a pillow from the elbow to the wrist. Check for adequate TMS coil access by holding the TMS coil against the patient&#39;s head. Make adjustments to the patient&#39;s position as required.</li>
	</ol>
	</li>
	<li>Position the patient in a chair or wheelchair for the test (alternative option).
	<ol>
		<li>Ensure the patient is sitting upright and comfortably in the chair. Place a pillow under each arm. Ensure the paretic forearm is pronated and fully supported by the pillow.</li>
	</ol>
	</li>
	<li>Check the EMG trace: Connect the cables between the patient and the EMG unit. Check to make sure the EMG signal is free from any electrical noise.</li>
	<li>Deliver TMS.
	<ol>
		<li>Two trained staff should be present for the TMS test. Instruct the patient to look straight ahead, keeping their head still and eyes open.</li>
		<li>The person holding the coil should stand next to the patient&#39;s head on their non-paretic side and position the center of the coil over the location of the primary motor cortex of the stroke-affected hemisphere. This is approximately 4 cm lateral from the vertex on the interaural line. 
		NOTE: Another way to identify the starting coil position is to measure approximately a third of the distance from the vertex to the front of the ear.</li>
		<li>Orient the coil with the handle pointing backwards, at an approximately 45&#176; angle in the midsagittal plane to produce a posterior-to-anterior current in the underlying tissue. 
		NOTE: The coil used in this protocol is a flat figure-eight coil, but a branding coil or circular coil can also be used.</li>
		<li>Adjust the bed height for the coil holder&#39;s comfort. Use a step if necessary. The second person (not the coil holder) is responsible for monitoring patient comfort throughout the TMS session. They may stand at the foot of the bed to monitor the patient and ensure the patient maintains a neutral head position or monitor the patient from the bedside while adjusting the TMS unit controls as necessary. 
		NOTE: This will depend on the individual TMS setup used. The patient should be monitored for comfort levels, alertness, and any adverse effects such as vasovagal responses to the TMS.</li>
		<li>Begin with a stimulus intensity of 30% maximum stimulator output (MSO). Increase intensity in 10% MSO steps with three to five stimuli at each intensity and scalp location.</li>
		<li>Move the coil systematically in 1 cm steps in each direction (anterior, posterior, medial, lateral) to find the optimal location for producing MEPs in the recorded muscles. Small adjustments to the coil rotation may also be necessary.</li>
		<li>Continue increasing the stimulus intensity and moving the coil until the MEPs are consistently observed in one or both muscles or until 100% MSO is reached.</li>
		<li>If 100% MSO is reached with no MEPs observed, use active facilitation to increase corticomotor excitability and the likelihood of eliciting a MEP. Ask the patient to hug a pillow to their chest with both arms, attempting to activate their paretic UL as much as possible. For patients with no distal UL activity, ask them to elevate and retract at the shoulder girdle.</li>
	</ol>
	</li>
	<li>Classify the MEP status of the patient.
	<ol>
		<li>Classify the patient as MEP+ if MEPs of any amplitude are observed with a consistent latency in response to at least five stimuli. This can be either at rest or during voluntary facilitation. FDI latencies are typically 20&#8211;30 ms while ECR latencies are typically 15&#8211;25 ms. MEPs do not have to exceed a peak-to-peak amplitude of 50 &#181;V.</li>
		<li>Classify the patient as MEP- if a MEP cannot be elicited at 100% MSO either at rest or while attempting voluntary facilitation.</li>
	</ol>
	</li>
	<li>Remove electrodes and wipe the skin with an alcohol wipe. The skin may be slightly red but this usually resolves without any treatment.</li>
</ol>

<ol>
	<li>Veerbeek, J. M., Kwakkel, G., van Wegen, E. E., Ket, J. C., Heymans, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+prediction+of+outcome+of+activities+of+daily+living+after+stroke:+a+systematic+review.">Early prediction of outcome of activities of daily living after stroke: a systematic review.</a> Stroke. 42 (5), 1482-1488 (2011).</li><li>Lohse, K. R., Schaefer, S. Y., Raikes, A. C., Boyd, L. A., Lang, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asking+New+Questions+with+Old+Data:+The+Centralized+Open-Access+Rehabilitation+Database+for+Stroke.">Asking New Questions with Old Data: The Centralized Open-Access Rehabilitation Database for Stroke.</a> Frontiers in Neurology. 7, 153 (2016).</li><li>Stinear, C., Ackerley, S., Byblow, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rehabilitation+is+initiated+early+after+stroke,+but+most+motor+rehabilitation+trials+are+not:+a+systematic+review.">Rehabilitation is initiated early after stroke, but most motor rehabilitation trials are not: a systematic review.</a> Stroke. 44 (7), 2039-2045 (2013).</li><li>Stinear, 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=Prediction+of+recovery+of+motor+function+after+stroke.">Prediction of recovery of motor function after stroke.</a> Lancet Neurology. 9 (12), 1228-1232 (2010).</li><li>Byblow, W. D., Stinear, C. M., Barber, P. A., Petoe, M. A., Ackerley, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proportional+recovery+after+stroke+depends+on+corticomotor+integrity.">Proportional recovery after stroke depends on corticomotor integrity.</a> Annals of Neurology. 78 (6), 848-859 (2015).</li><li>Stinear, 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=Prediction+of+motor+recovery+after+stroke:+advances+in+biomarkers.">Prediction of motor recovery after stroke: advances in biomarkers.</a> Lancet Neurology. 16 (10), 826-836 (2017).</li><li>Kim, B., Winstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+Neurological+Biomarkers+of+Brain+Impairment+Be+Used+to+Predict+Poststroke+Motor+Recovery?+A+Systematic+Review.">Can Neurological Biomarkers of Brain Impairment Be Used to Predict Poststroke Motor Recovery? A Systematic Review.</a> Neurorehabilitation and Neural Repair. 31 (1), 3-24 (2016).</li><li>Boyd, 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=Biomarkers+of+stroke+recovery:+Consensus-based+core+recommendations+from+the+Stroke+Recovery+and+Rehabilitation+Roundtable.">Biomarkers of stroke recovery: Consensus-based core recommendations from the Stroke Recovery and Rehabilitation Roundtable.</a> International Journal of Stroke. 12 (5), 480-493 (2017).</li><li>Escudero, J. V., Sancho, J., Bautista, D., Escudero, M., Lopez-Trigo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+value+of+motor+evoked+potential+obtained+by+transcranial+magnetic+brain+stimulation+in+motor+function+recovery+in+patients+with+acute+ischemic+stroke.">Prognostic value of motor evoked potential obtained by transcranial magnetic brain stimulation in motor function recovery in patients with acute ischemic stroke.</a> Stroke. 29 (9), 1854-1859 (1998).</li><li>Pennisi, G., 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=Absence+of+response+to+early+transcranial+magnetic+stimulation+in+ischemic+stroke+patients:+prognostic+value+for+hand+motor+recovery.">Absence of response to early transcranial magnetic stimulation in ischemic stroke patients: prognostic value for hand motor recovery.</a> Stroke. 30 (12), 2666-2670 (1999).</li><li>Rapisarda, G., Bastings, E., de Noordhout, A. M., Pennisi, G., Delwaide, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+motor+recovery+in+stroke+patients+be+predicted+by+early+transcranial+magnetic+stimulation?.">Can motor recovery in stroke patients be predicted by early transcranial magnetic stimulation?.</a> Stroke. 27 (12), 2191-2196 (1996).</li><li>Bembenek, J. P., Kurczych, K., Karli Nski, M., Czlonkowska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prognostic+value+of+motor-evoked+potentials+in+motor+recovery+and+functional+outcome+after+stroke+-+a+systematic+review+of+the+literature.">The prognostic value of motor-evoked potentials in motor recovery and functional outcome after stroke - a systematic review of the literature.</a> Functional Neurology. 27 (2), 79-84 (2012).</li><li>Smania, N., 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=Active+finger+extension:+a+simple+movement+predicting+recovery+of+arm+function+in+patients+with+acute+stroke.">Active finger extension: a simple movement predicting recovery of arm function in patients with acute stroke.</a> Stroke. 38 (3), 1088-1090 (2007).</li><li>Nijland, R. H., van Wegen, E. E., Harmeling-van der Wel, B. C., Kwakkel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EPOS+Investigators.+Presence+of+finger+extension+and+shoulder+abduction+within+72+hours+after+stroke+predicts+functional+recovery:+early+prediction+of+functional+outcome+after+stroke:+the+EPOS+cohort+study.">EPOS Investigators. Presence of finger extension and shoulder abduction within 72 hours after stroke predicts functional recovery: early prediction of functional outcome after stroke: the EPOS cohort study.</a> Stroke. 41 (4), 745-750 (2010).</li><li>Katrak, P., 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=Predicting+upper+limb+recovery+after+stroke:+the+place+of+early+shoulder+and+hand+movement.">Predicting upper limb recovery after stroke: the place of early shoulder and hand movement.</a> Archives of Physical Medicine and Rehabilitation. 79 (7), 758-761 (1998).</li><li>Stinear, C. M., Barber, P. A., Petoe, M., Anwar, S., Byblow, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PREP+algorithm+predicts+potential+for+upper+limb+recovery+after+stroke.">The PREP algorithm predicts potential for upper limb recovery after stroke.</a> Brain. 135 (Pt 8), 2527-2535 (2012).</li><li>Stinear, C. 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=PREP2:+A+biomarker-based+algorithm+for+predicting+upper+limb+function+after+stroke.">PREP2: A biomarker-based algorithm for predicting upper limb function after stroke.</a> Annals of Clinical and Translational Neurology. 4 (11), 811-820 (2017).</li><li>Groppa, S., 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+practical+guide+to+diagnostic+transcranial+magnetic+stimulation:+report+of+an+IFCN+committee.">A practical guide to diagnostic transcranial magnetic stimulation: report of an IFCN committee.</a> Clinical Neurophysiology. 123 (5), 858-882 (2012).</li><li>Barker, A. T., Jalinous, R., Freeston, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+magnetic+stimulation+of+human+motor+cortex.">Non-invasive magnetic stimulation of human motor cortex.</a> Lancet. 1 (8437), 1106-1107 (1985).</li><li>Stinear, C. M., Byblow, W. D., Ackerley, S. J., Barber, P. A., Smith, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+Recovery+Potential+for+Individual+Stroke+Patients+Increases+Rehabilitation+Efficiency.">Predicting Recovery Potential for Individual Stroke Patients Increases Rehabilitation Efficiency.</a> Stroke. 48 (4), 1011-1019 (2017).</li><li>Connell, L. A., Smith, M. C., Byblow, W. D., Stinear, 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=Implementing+biomarkers+to+predict+motor+recovery+after+stroke.">Implementing biomarkers to predict motor recovery after stroke.</a> NeuroRehabilitation. 43 (1), 41-50 (2018).</li><li>Smith, M. C., Ackerley, S. J., Barber, P. A., Byblow, W. D., Stinear, 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=PREP2+Algorithm+Predictions+Are+Correct+at+2+Years+Poststroke+for+Most+Patients.">PREP2 Algorithm Predictions Are Correct at 2 Years Poststroke for Most Patients.</a> Neurorehabilitation and Neural Repair. 33 (8), 635-642 (2019).</li><li>Rossi, S., Hallett, M., Rossini, P. M., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety,+ethical+considerations,+and+application+guidelines+for+the+use+of+transcranial+magnetic+stimulation+in+clinical+practice+and+research.">Safety, ethical considerations, and application guidelines for the use of transcranial magnetic stimulation in clinical practice and research.</a> Clinical Neurophysiology. 120 (12), 2008-2039 (2009).</li><li>Talelli, P., Greenwood, R. J., Rothwell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arm+function+after+stroke:+neurophysiological+correlates+and+recovery+mechanisms+assessed+by+transcranial+magnetic+stimulation.">Arm function after stroke: neurophysiological correlates and recovery mechanisms assessed by transcranial magnetic stimulation.</a> Clinical Neurophysiology. 117 (8), 1641-1659 (2006).</li></ol>

The authors have nothing to disclose.

CST function evaluated with MEP status is a key prognostic biomarker for UL recovery and outcome after stroke. A total of 95% of patients with a functional CST at 1 week poststroke achieve an Action Research Arm Test (ARAT) score of at least 34 out of 57 by 3 months poststroke17. Conversely, 100% of patients without a functional CST at 1 week poststroke achieve an ARAT score of less than 34 by 3 months poststroke17. Evaluating CST function within a week poststroke may impro...

Upper limb function is commonly impaired after stroke, and recovery of UL function is important for regaining independence in daily living activities1. Stroke rehabilitation trials are often aimed at improving UL recovery and outcomes after stroke. The majority of stroke rehabilitation research is conducted with patients at the chronic stage (&#62;6 months poststroke), yet most rehabilitation occurs early after stroke2,3. More research needs to be conducted with patients soon after a stroke to build an evidence base for rehabilitation practice.
One of the greatest challenges when conducting research soon after the stroke is detecting the effects of the intervention against the background of recovery occurring during the initial weeks and months after the stroke. High intersubject variability in clinical presentation and recovery creates noise that can obscure the beneficial effects of interventions. Intervention and control groups are typically balanced on clinical measures of initial neurological impairment. However, these measures are often poor predictors of the patient&#39;s potential for subsequent recovery, particularly those with severe initial impairment4,5. This means that groups can be matched for baseline clinical measures and not matched for their recovery potential, which makes it more difficult to ascertain the intervention&#39;s effects. Biomarkers can address this challenge by identifying an individual patient&#39;s potential for motor recovery, so that groups can be accurately matched and stratified6,7,8. Biomarkers can also be used to select patients who are most likely to respond to the intervention&#39;s known or hypothesized mechanisms of action6.
The functional integrity of the corticospinal tract (CST) is a key biomarker that predicts recovery of UL function after stroke5,8,9,10,11,12. The CST conveys descending motor output from the primary motor cortex to the spinal cord and is essential for coordination and fine motor control. Patients with a functional CST after stroke are more likely to regain strength, coordination, and dexterity than patients without. A clinical assessment can be sufficient to confirm that the CST is functional in mildly impaired patients13,14,15. However, patients with more severe initial impairment may or may not have a functional CST, and a neurophysiological assessment using transcranial magnetic stimulation (TMS) is needed9,10,11,16,17.
TMS is a noninvasive and painless technique that can be used to test CST function18. The TMS coil delivers a magnetic stimulus over the primary motor cortex that generates a descending volley in the CST, eliciting a motor-evoked potential (MEP) in the muscles of the contralateral limb19. The presence of a MEP in the paretic arm or hand (MEP+) indicates a functional CST and is associated with greater potential for recovery of UL function. Patients who are MEP- are most likely to have worse UL recovery, with no return of coordinated and dexterous hand function4,6,9,12,16.
Testing all patients with TMS is impractical and unnecessary, as those with mild initial impairment most likely have a functional CST17. Therefore, a hierarchical approach is needed so that TMS is only used for patients with more severe initial impairment. The PREP2 algorithm was developed using a combination of clinical measures and TMS to evaluate CST function and predict likely UL outcome at 3 months poststroke (Figure 1)17. PREP2 starts at day 3 poststroke by testing the strength of shoulder abduction and finger extension in the paretic arm (SAFE score), using Medical Research Council grades. If the sum of these grades is 5 or more out of 10, it is &#34;safe&#34; to assume the patient is MEP+. These patients are expected to have a good or excellent UL outcome by 3 months poststroke, depending on their age17. These patients do not need TMS to determine MEP status, minimizing cost and unnecessary testing for the patient.
Patients with a SAFE score of less than 5 on day 3 poststroke require TMS to determine the functional integrity of their CST. If a MEP can be elicited from the paretic extensor carpi radialis (ECR) or first dorsal interosseus (FDI) muscles, the patient is MEP+ and is expected to recover fine motor control of the hand by 3 months poststroke. Approximately half of patients with a SAFE score less than 5 on day 3 poststroke are MEP+. Importantly, patients can have a SAFE score as low as zero and be MEP+. This illustrates the need for TMS in this subgroup of patients, as clinical assessment alone cannot distinguish between patients with and without a functional CST. Patients who are MEP- have significant CST damage. These patients are expected to have a limited or poor UL functional outcome depending on their overall stroke severity, measured with the National Institute of Health Stroke Scale (NIHSS) (Figure 1)17. These MEP- patients are not expected to regain coordinated and dexterous finger control and can be grouped together for research purposes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60665/60665fig01.jpg" /> 
Figure 1: The PREP2 algorithm. SAFE = Shoulder Abduction, Finger Extension score, which is the sum of the Medical Research Council grades for each of these movements out of 5, for a total SAFE score out of 10. MEP+ = Motor Evoked Potentials can be elicited from the paretic extensor carpi radialis (ECR) and/or first dorsal interosseous (FDI) muscles of the paretic UL using transcranial magnetic stimulation. NIHSS = National Institutes of Health Stroke Scale. The algorithm predicts one of four possible UL functional outcomes at 3 months poststroke. Each prediction category is associated with a rehabilitation focus that can be used to tailor UL therapy2. The colored dots represent, proportionally, PREP2 algorithm accuracy. The dots are color-coded based on the outcome category actually achieved 3 months poststroke (Green = Excellent; Blue = Good; Orange = Limited; Red = Poor). Figure reproduced from Stinear et al.17. <a href="https://www.jove.com/files/ftp_upload/60665/60665fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In clinical practice, PREP2 predicts one of four outcome categories that can be used to tailor rehabilitation for individual patients and help patients and families to understand what they can expect for their UL recovery. To date, PREP2 is the only externally validated UL prediction tool that combines clinical assessment and biomarker information in a decision tree17. It is also the only UL prediction tool with research on the effects of implementation in clinical practice20,21. PREP2 predictions are accurate for about 75% of patients, too optimistic for 17% and too pessimistic for 8% of patients at 3 months poststroke17. Accuracy is highest for MEP- patients (accurate for 90% of MEP- patients), highlighting the value of using TMS to identify these patients with severe damage to the descending motor pathways17. PREP2 remains correct for around 80% of patients at 2 years poststroke22. This supports the use of PREP2 to predict UL functional motor outcomes at 3 months and longer term. Information about delivering PREP2 predictions and using them in clinical practice is outside the scope of this methods paper, but detailed resources are available online23.
PREP2 provides researchers with a tool to select and stratify patients for clinical trials. This allows patients to be grouped not only according to baseline clinical characteristics, but also their neurobiological potential for UL recovery. Despite the mounting evidence for the use of TMS as a prognostic biomarker for UL recovery, lack of familiarity with TMS protocols in hospital settings with subacute stroke patients may be a barrier to its use in research. Therefore, this protocol aims to demonstrate how to use the SAFE score and TMS to evaluate CST function in patients in a hospital setting early after stroke.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>alcohol/skin cleansing wipes</td>
			<td>Reynard</td>
			<td></td>
			<td>alcohol prep pads</td>
		</tr>
		<tr>
			<td>electromyography electrodes</td>
			<td>3M</td>
			<td></td>
			<td>red dot electrodes</td>
		</tr>
		<tr>
			<td>Magstim TMS coil</td>
			<td>Magstim</td>
			<td></td>
			<td>flat figure-8 coil</td>
		</tr>
		<tr>
			<td>razors</td>
			<td>any</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>skin prep tape</td>
			<td>3M</td>
			<td></td>
			<td>red dot skin prep tape</td>
		</tr>
		<tr>
			<td>TMS stimulator</td>
			<td>Magstim</td>
			<td></td>
			<td>Magstim 200 single pulse stimulator</td>
		</tr>
	</tbody>
</table>

determining the functional status of the corticospinal tract within one week of stroke

High interindividual variability in the recovery of upper limb (UL) function after stroke means it is difficult to predict an individual&#39;s potential for recovery based on clinical assessments alone. The functional integrity of the corticospinal tract is an important prognostic biomarker for recovery of UL function, particularly for those with severe initial UL impairment. This article presents a protocol for evaluating corticospinal tract function within 1 week of stroke. This protocol can be used to select and stratify patients in trials of interventions designed to improve UL motor recovery and outcomes after stroke. The protocol also forms part of the PREP2 algorithm, which predicts UL function for individual patients 3 months poststroke. The algorithm sequentially combines a UL strength assessment, age, transcranial magnetic stimulation, and stroke severity, within a few days of the stroke. The benefits of using PREP2 in clinical practice are described elsewhere. This article focuses on the use of a UL strength assessment and transcranial magnetic stimulation to evaluate corticospinal tract function.

The SAFE score and TMS can be used to ascertain the functional status of the CST within one week of stroke. Patients who have a SAFE score of at least 5 on day 3, or are MEP+ when tested with TMS, have a functional CST and are expected to regain at least some coordination and dexterity. Patients who are MEP- do not have a functional CST and therefore are likely to be limited to improvements in proximal arm movements and gross movements of the hand. The functional status of the CST can therefore be used to select patients...

Watch this Scientific Journal Video about Determining the Functional Status of the Corticospinal Tract Within One Week of Stroke at JoVE.com

Determining the Functional Status of the Corticospinal Tract Within One Week of Stroke

This protocol is for evaluating corticospinal tract function within 1 week of stroke. It can be used to select and stratify patients in trials of interventions designed to improve upper limb motor recovery and outcomes and in clinical practice for predicting upper limb functional outcomes 3 months after stroke.

High interindividual variability in the recovery of upper limb (UL) function after stroke means it is difficult to predict an individual&#39;s potential ...

determining-functional-status-corticospinal-tract-within-one-week

Research

JoVE Journal

Neuroscience

8.4K Views.  University of Auckland. This protocol is for evaluating corticospinal tract function within 1 week of stroke. It can be used to select and stratify patients in trials of interventions designed to improve upper limb motor recovery and outcomes and in clinical practice for predicting upper limb functional outcomes 3 months after stroke.

Video: Determining the Functional Status of the Corticospinal Tract Within One Week of Stroke

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has great importance for distal muscle control in humans and, to a lesser extent, in rodents. A unilateral cut of one pyramid results in loss of CST innervation of the spinal cord mainly on the contralateral side of the spinal cord leading to transient motor disability in the forelimbs and sustained loss of dexterity. Ipsilateral projections of the corticospinal tract are minor. We have refined our surgical method to increase the chances of lesion completeness. We describe postsurgical care. Deficits on the Montoya staircase pellet reaching test and the horizontal ladder test shown here are detected up to 8 weeks postinjury. Deficits on the cylinder rearing test are only detected transiently. Therefore, the cylinder test may only be suitable for detection of short term recovery. We show how, electrophysiologically and anatomically, one may assess lesions and plastic changes. We also describe how to analyse fibers from the uninjured CST sprouting across the midline into the deprived areas. It is challenging to obtain &#62;90% complete lesions consistently due to the proximity to the basilar artery in the medulla oblongata and survival rates can be low. Alternative surgical approaches and behavioural testing are described in this protocol. The pyramidotomy model is a good tool for assessing neuroplasticity-inducing treatments, which increase sprouting of intact fibers after injury.

The corticospinal tract, one of the major sensorimotor tracts, can be lesioned unilaterally in the rodent brainstem in order to test neuroplasticity-inducing therapies for the central nervous system. This surgical procedure (&#8220;pyramidotomy&#8221;) and postoperative assessments are described in this protocol.

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has ...

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.
In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. 
This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP protein.

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a ...

Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans and can be assessed using transcranial magnetic stimulation (TMS). Given the role of distal leg muscles in upright postural and dynamic tasks, such as walking, a growing research interest in the assessment and modulation of the corticospinal tracts relative to the function of these muscles has emerged in the last decade. However, methodological parameters used in previous work have varied across studies making the interpretation of results from cross-sectional and longitudinal studies less robust. Therefore, use of a standardized TMS protocol specific to the assessment of leg muscles&#39; corticomotor response (CMR) will allow for direct comparison of results across studies and cohorts. The objective of this paper is to present a protocol that provides the flexibility to simultaneously assess the bilateral CMR of two main ankle antagonistic muscles, the tibialis anterior and soleus, using single pulse TMS with a neuronavigation system. The present protocol is applicable while the examined muscle is either fully relaxed or isometrically contracted at a defined percentage of maximum isometric voluntary contraction. Using each subject&#39;s structural MRI with the neuronavigation system ensures accurate and precise positioning of the coil over the leg cortical representations during assessment. Given the inconsistency in CMR derived measures, this protocol also describes a standardized calculation of these measures using automated algorithms. Though this protocol is not conducted during upright postural or dynamic tasks, it&#160;can be used to assess bilaterally any pair of leg muscles, either antagonistic or synergistic, in both neurologically intact and impaired subjects.

The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system.

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans ...

Using Zebrafish Larvae to Study the Pathological Consequences of Hemorrhagic Stroke

Despite being the most severe subtype of stroke with high global mortality, there is no specific treatment for patients with intracerebral hemorrhage (ICH). Modelling ICH pre-clinically has proven difficult, and current rodent models poorly recapitulate the spontaneous nature of human ICH. Therefore, there is an urgent requirement for alternative pre-clinical methodologies for study of disease mechanisms in ICH and for potential drug discovery.
The use of zebrafish represents an increasingly popular approach for translational research, primarily due to a number of advantages they possess over mammalian models of disease, including prolific reproduction rates and larval transparency allowing for live imaging. Other groups have established that zebrafish larvae can exhibit spontaneous ICH following genetic or chemical disruption of cerebrovascular development. The aim of this methodology is to utilize such models to study the pathological consequences of brain hemorrhage, in the context of pre-clinical ICH research. By using live imaging and motility assays, brain damage, neuroinflammation and locomotor function following ICH can be assessed and quantified.
This study shows that key pathological consequences of brain hemorrhage in humans are conserved in zebrafish larvae highlighting the model organism as a valuable in vivo&#160;system for pre-clinical investigation of ICH. The aim of this methodology is to enable the pre-clinical stroke community to employ the zebrafish larval model as an alternative complementary model system to rodents.

Here we present a protocol to quantify brain injury, locomotor deficits and neuroinflammation following bleeding in the brain in zebrafish larvae, in the context of human intracerebral hemorrhage (ICH).

Despite being the most severe subtype of stroke with high global mortality, there is no specific treatment for patients with intracerebral hemorrhage ...

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

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 ...