The authors would like to acknowledge Ms. Angela Wirsching, who was a key contributor to the research we cite in the representative results section.

The authors verify that the Indiana University Institutional Review Board approved the study (protocol # 1610743422) and written informed consent was obtained.
NOTE: The SSHM was integrated into a repeated measures design that is intended to investigate changes among dependent variables within subjects at 0 h, 2 h, and 24 h post-intervention compared to individual pre-intervention values. This study design allows researchers to track changes for a 24 h period, which is the typical timeframe between athletic practices. In the present study, soccer players were randomly assigned to either a soccer heading (n = 18) or a soccer kicking group (n = 16).
1. Setup
<ol>
	<li>Following baseline measurement collection (pre-intervention), begin the SSHM with the positioning of a soccer ball launcher approximately 40 ft away from the subject, as well as ensuring the soccer ball is inflated to 9 psi.</li>
	<li>The face of the machine displays two identical dials, which regulate the speed of the left and right wheels, and an on/off switch in between them. Set both these dials to a standardized speed of choice. 
	NOTE: For this demonstration purpose, the ball traveling speed has been set to 30 mph. This speed was chosen as it simulates a soccer throw-in from the sideline to midfield. Soccer players frequently perform this maneuver during practice and games.</li>
	<li>Place 3 inch blocks underneath the wheels of the ball launcher to allow for the desired trajectory (no blocks needed for kicking subjects). Once this is complete, the ball launcher is then angled to 40&#176;, which is measured as the angle between the ground and the midline of the rotating wheels. 
	NOTE: The angle measurement is taken with a goniometer, and the machine can be adjusted after loosening a knob located along the blue rails where the soccer ball is loaded.</li>
	<li>Once the soccer machine is properly set, fit the subjects with a triaxial accelerometer embedded within a head-band pocket and positioned directly below the external occipital protuberance (inion) to monitor linear and rotational head accelerations.</li>
	<li>Start up the corresponding software for the accelerometer, and enter the subject&#8217;s information accordingly. At this point, the subject is ready to begin the familiarization trials of the intervention (heading, kicking).</li>
</ol>
2. Familiarization trials
<ol>
	<li>Position the subject approximately 40 ft in front of the ball launcher.</li>
	<li>Be sure to explain to the subject that the ball launcher will volley the soccer ball to them and that they simply need to simulate intervention contact with the ball (i.e., heading subjects will catch the ball with their hands in front of their forehead before head-to-ball contact is made, kicking subjects will &#8220;trap&#8221; the ball on the ground with their foot instead of volleying the ball back, and standing subjects will remain static and not make contact with the ball).</li>
	<li>When the subject understands and feels ready, have the researcher turn the ball launcher on, load the soccer ball onto the blue rails, and finally push the ball into the rotating wheels after a 3-2-1 countdown.</li>
	<li>After stopping the ball as described previously (step 2.2), have the subject roll the ball back.</li>
	<li>Repeat steps 2.3 and 2.4 two to four additional times (no rest time required between) to ensure that the subject positioning is correct and interaction with the ball will be safe and controlled. This concludes the familiarization trials.</li>
</ol>
3. Intervention
<ol>
	<li>Verbally confirm that the subject is ready. Once confirmed, give instructions to the heading subjects to only make forehead contact with the ball; tell the subjects to avoid potential impacts to the crown, parietal, and temporal lobes. Instruct the kicking subjects to kick the ball only while it is in flight as ball contact with the ground will attenuate the subsequent impact to the foot.</li>
	<li>Instruct both heading and kicking subjects to volley the ball to a target (additional researcher) approximately half the distance between them and the machine. As best they can, ask subjects to do this in a manner that will mimic the arched trajectory the ball took during its flight toward the subject.</li>
	<li>Activate the triaxial accelerometer and begin the recording.</li>
	<li>Load the soccer ball onto the blue rails, push the ball into the rotating wheels after a 3-2-1 countdown, and make sure appropriate contact is made.</li>
	<li>Repeat step 3.4 nine more times with a 60 s rest in between bouts. If the subject forgoes interaction with the ball (due to inopportune placement or suspected contact with the body in an area that must be avoided), then volley the ball to the subject again promptly, without any rest period.</li>
	<li>In between each head contact with the ball, verify that the triaxial accelerometer registered an impact (using triaxial software; kicking subjects should not register G-force).</li>
	<li>Once the intervention is concluded, turn off the ball launcher and stop the triaxial recording (important, as the movement required to remove the headband could record another &#8220;impact&#8221;). Once the recording has stopped, remove the headband.</li>
</ol>

<ol>
	<li>Goldstein, L. E., 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=Chronic+traumatic+encephalopathy+in+blast-exposed+military+veterans+and+a+blast+neurotrauma+mouse+model.">Chronic traumatic encephalopathy in blast-exposed military veterans and a blast neurotrauma mouse model.</a> Science Translational Medicine. 4 (134), 134ra60 (2012).</li><li>Kraus, M. F., 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=White+matter+integrity+and+cognition+in+chronic+traumatic+brain+injury:+a+diffusion+tensor+imaging+study.">White matter integrity and cognition in chronic traumatic brain injury: a diffusion tensor imaging study.</a> Brain: A Journal of Neurology. 130 (Pt 10), 2508-2519 (2007).</li><li>McKee, A. C., 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=The+spectrum+of+disease+in+chronic+traumatic+encephalopathy.">The spectrum of disease in chronic traumatic encephalopathy.</a> Brain: A Journal of Neurology. 136 (Pt. 136 (Pt 1), 43-64 (2013).</li><li>Mez, J., 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=Clinicopathological+Evaluation+of+Chronic+Traumatic+Encephalopathy+in+Players+of+American+Football.">Clinicopathological Evaluation of Chronic Traumatic Encephalopathy in Players of American Football.</a> JAMA. 318 (4), 360-370 (2017).</li><li>Tagge, C. 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=Concussion,+microvascular+injury,+and+early+tauopathy+in+young+athletes+after+impact+head+injury+and+an+impact+concussion+mouse+model.">Concussion, microvascular injury, and early tauopathy in young athletes after impact head injury and an impact concussion mouse model.</a> Brain: A Journal of Neurology. 141 (2), 422-458 (2018).</li><li>Bailes, J. E., Petraglia, A. L., Omalu, B. I., Nauman, E., Talavage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+subconcussion+in+repetitive+mild+traumatic+brain+injury.">Role of subconcussion in repetitive mild traumatic brain injury.</a> Journal of Neurosurgery. 119 (5), 1235-1245 (2013).</li><li>Schnebel, B., Gwin, J. T., Anderson, S., Gatlin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+study+of+head+impacts+in+football:+a+comparison+of+National+Collegiate+Athletic+Association+Division+I+versus+high+school+impacts.">In vivo study of head impacts in football: a comparison of National Collegiate Athletic Association Division I versus high school impacts.</a> Neurosurgery. 60 (3), 490-496 (2007).</li><li>Guskiewicz, K. 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=Measurement+of+head+impacts+in+collegiate+football+players:+relationship+between+head+impact+biomechanics+and+acute+clinical+outcome+after+concussion.">Measurement of head impacts in collegiate football players: relationship between head impact biomechanics and acute clinical outcome after concussion.</a> Neurosurgery. 61 (6), 1244-1253 (2007).</li><li>Combat veterans coming home with CTE. CBS News Available from: <a target="_blank" href="https://www.cbsnews.com/news/60-minutes-combat-veterans-coming-home-with-cte-brain-injury/">https://www.cbsnews.com/news/60-minutes-combat-veterans-coming-home-with-cte-brain-injury/</a> (2018)</li><li>Ward, J., Williams, J., Manchester, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=111+N.F.L.+Brains.+All+But+One+Had+C.T.E.">111 N.F.L. Brains. All But One Had C.T.E.</a> The New York Times. , (2017).</li><li>Ling, H., 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=Mixed+pathologies+including+chronic+traumatic+encephalopathy+account+for+dementia+in+retired+association+football+(soccer)+players.">Mixed pathologies including chronic traumatic encephalopathy account for dementia in retired association football (soccer) players.</a> Acta Neuropathologica. 133 (3), 337-352 (2017).</li><li>Kawata, K., Tierney, R., Phillips, J., Jeka, J. J. <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+Repetitive+Sub-concussive+Head+Impacts+on+Ocular+Near+Point+of+Convergence.">Effect of Repetitive Sub-concussive Head Impacts on Ocular Near Point of Convergence.</a> International Journal of Sports Medicine. 37 (5), 405-410 (2016).</li><li>Wirsching, A., Chen, Z., Bevilacqua, Z. W., Huibregtse, M. E., Kawata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Acute+Increase+in+Plasma+Neurofilament+Light+with+Repetitive+Subconcussive+Head+Impacts:+A+Pilot+Randomized+Control+Trial.">Association of Acute Increase in Plasma Neurofilament Light with Repetitive Subconcussive Head Impacts: A Pilot Randomized Control Trial.</a> Journal of Neurotrauma. , (2018).</li><li>Coon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Acute Effects of Sleep Deprivation on Ocular-Motor Function as Assessed by King-Devick Test Performance. , (2018).</li><li>Hwang, S., Ma, L., Kawata, K., Tierney, R., Jeka, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vestibular+Dysfunction+after+Subconcussive+Head+Impact.">Vestibular Dysfunction after Subconcussive Head Impact.</a> Journal of Neurotrauma. 34 (1), 8-15 (2017).</li><li>Oliver, J. 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=Serum+Neurofilament+Light+in+American+Football+Athletes+over+the+Course+of+a+Season.">Serum Neurofilament Light in American Football Athletes over the Course of a Season.</a> Journal of Neurotrauma. 33 (19), 1784-1789 (2016).</li><li>Shahim, P., Zetterberg, H., Tegner, Y., Blennow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+neurofilament+light+as+a+biomarker+for+mild+traumatic+brain+injury+in+contact+sports.">Serum neurofilament light as a biomarker for mild traumatic brain injury in contact sports.</a> Neurology. 88 (19), 1788-1794 (2017).</li><li>FIFA Communications Division, Information Services. FIFA Big Count 2006: 270 million people active in football Available from: <a target="_blank" href="https://www.fifa.com/mm/document/fifafacts/bcoffsurv/bigcount.statspackage_7024.pdf">https://www.fifa.com/mm/document/fifafacts/bcoffsurv/bigcount.statspackage_7024.pdf</a> (2007)</li><li>Dorminy, 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+soccer+heading+ball+speed+on+S100B,+sideline+concussion+assessments+and+head+impact+kinematics.">Effect of soccer heading ball speed on S100B, sideline concussion assessments and head impact kinematics.</a> Brain Injury. , 1-7 (2015).</li><li>Bretzin, A. C., Mansell, J. L., Tierney, R. T., McDevitt, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+Differences+in+Anthropometrics+and+Heading+Kinematics+Among+Division+I+Soccer+Athletes.">Sex Differences in Anthropometrics and Heading Kinematics Among Division I Soccer Athletes.</a> Sports Health. 9 (2), 168-173 (2017).</li><li>Crisco, J. J., 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=Frequency+and+location+of+head+impact+exposures+in+individual+collegiate+football+players.">Frequency and location of head impact exposures in individual collegiate football players.</a> Journal of Athletic Training. 45 (6), 549-559 (2010).</li><li>Duma, S. 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=Analysis+of+real-time+head+accelerations+in+collegiate+football+players.">Analysis of real-time head accelerations in collegiate football players.</a> Clinical Journal of Sport Medicine. 15 (1), 3-8 (2005).</li><li>Reynolds, B. B., 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=Practice+type+effects+on+head+impact+in+collegiate+football.">Practice type effects on head impact in collegiate football.</a> Journal of Neurosurgery. , 1-10 (2015).</li><li>Kawata, K., 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=Association+of+Football+Subconcussive+Head+Impacts+With+Ocular+Near+Point+of+Convergence.">Association of Football Subconcussive Head Impacts With Ocular Near Point of Convergence.</a> JAMA Ophthalmology. 134 (7), 763-769 (2016).</li><li>Wilcox, B. J., 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=Head+impact+exposure+in+male+and+female+collegiate+ice+hockey+players.">Head impact exposure in male and female collegiate ice hockey players.</a> Journal of Biomechanics. 47 (1), 109-114 (2014).</li><li>Wallace, C., 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=Heading+in+soccer+increases+serum+neurofilament+light+protein+and+SCAT3+symptom+metrics.">Heading in soccer increases serum neurofilament light protein and SCAT3 symptom metrics.</a> BMJ Open Sport &amp; Exercise Medicine. 4 (1), e000433 (2018).</li></ol>

The authors have nothing to disclose.

While contact sports like American football might appear to be driving the need for a concise research model to study subconcussive impacts, other sports like soccer may account for the dominant share of subconcussive exposure around the globe as approximately 265 million people participate in what is perhaps the world&#8217;s most popular sport19. However, while the majority of the suspected long-term neurodegenerative effects of subconcussion have been autopsied in American football players, the resemblance of football head impacts to soccer headers is surprisingly large. For instance, 10 bouts of soccer heading in the current study induced approximately 300 g and 35 krad/s2, which were near identical to previous reports using similar subconcussion models20,21. These impact kinematics were comparable to the hits observed in American football, where the average college football player incurs 7.0-9.4 hits during practice and 25 hits during games, with a mean peak linear acceleration per hit of 28.8-32.0 g22,23,24,25. Furthermore, other contact sports, such as ice hockey, have displayed similar or greater peak linear head accelerations than these results. For example, a recent study examining both male and female collegiate ice hockey athletes revealed that, over the course of a season, these athletes would register peak linear accelerations of 41.6 g (36.6-49.5 g) and 40.8 g (36.5-49.9 g) for males and females, respectively (median [IQR])26. Additionally, the head impact telemetry system that was utilized in this ice hockey study revealed that nearly one-third of males (28.0% [21.2%-33.5%]) and females (29.3% [24.8%-32.1%]) (median [IQR]) were inflicted on the front of the head26. These results directly parallel to the head impacts induced by using the SSHM, which indicates the vast clinical utility of the current methodology.
There is, however, one limitation that should be addressed when attempting to extrapolate soccer and American football head impacts to other contact sports like ice hockey. Head impacts in ice hockey are not routine drills and are penalized, and they usually are the result of deliberate contact with another player. In fact, subconcussive head impact incidence for collegiate ice hockey athletes is known to be substantially low: 1.3 (1.0-1.7) per practice and 6.3 (3.5-9.0) per game for males and 0.9 (0.6-1.0) per practice and 3.7 (2.5-4.9) per game for females26.
The present study is not the first to explore the expression of NF-L following head trauma; however, it is the first to isolate head trauma as the cause for NF-L expression outside the central nervous system. Oliver et al. assessed for serum NF-L expression within American football athletes at eight different time points over the course of a season (~6 months)17. Players were categorized into two groups, starters and nonstarters, with an assumption that starter athletes will be exposed to larger quantities of head hits, whereas nonstarters will incur lower quantities of head hits than the starters. The authors identified significant increases in serum NF-L expressions in the starter group over time across the season17. Conversely, the nonstarter group remained consistent throughout the season. Similarly, Shahim et al. analyzed serum NF-L levels in boxers following a subacute phase of subconcussive head trauma (7-10 days prior)18. In their analysis, boxers were categorized into two groups (mild vs. severe head impact groups) based on an anecdotal threshold of &#60;16 vs. &#8805;16 head hits during a boxing match, respectively. The serum expression of NF-L was significantly elevated in the severe head impact group when compared to the mild impact group, suggesting a dose-dependent response to head impact and biomarker expression18. While the presented literature elicits strong reasoning for NF-L as a marker of axonal injury, neither study exhibits robust control of confounding variables seen throughout sport (thermogenesis, hydration status, body contacts, head kinematics, etc.). This gap in methodology underpins the need for the SSHM. By using the SSHM, Wirsching et al.14 and Wallace et al.27 were able to control aforementioned confounding variables, coupled with the monitoring location, magnitude, and quantity of head impacts, and study the head impact dose-response profile of NF-L levels in the blood. Therefore, the SSHM provides safe and standardized means to study the effect of subconcussive head impacts and validate clinical findings.
The SSHM shows promise as being a relevant, reproducible, and leading method of isolating and examining the effect of subconcussive head impacts irrespective of sport; however, there are a few limitations to consider when adopting this method. First, the SSHM has a strong ability to control for environmental covariates (previously described); however, this means that a temperature-controlled indoor facility is required. Second, in an effort to ensure safe and controlled head contacts, we set participant exclusion criteria to a minimum of 5 years of soccer heading experience. This cutoff was used to eliminate potential injurious head contacts due to novice heading technique. Finally, this protocol requires specific equipment and multiple researchers to conduct, which may not be accessible unanimously.
The SSHM provides an invaluable modality for researchers to confidently validate results found in field studies. This confidence stems from the ability of the SSHM to control for both internal and external confounding factors such as impact location and quantity, body damage, exercise effect, and perspiration, to mention a few. Furthermore, because the SSHM has shown similar head impact magnitudes as other sports like ice hockey and American football, the claim can be made that this methodology bears vast clinical relevance not only to soccer athletes but also to American football, ice hockey, rugby, and boxing athletes. Lastly, because the SSHM does not segregate populations, as do some unisex sports like American football, all genders and ethnicities can be studied.

Long-term, repetitive exposure to subconcussive head impacts has been proposed as one of the key contributors for developing the neurodegenerative disease CTE1,2,3,4,5. Each year, approximately 2.5 million high school and college athletes engage in contact sports that frequently induce these subconcussive insults through rapid acceleration-deceleration of the body and head6,7. Specifically, contact sport athletes may experience several 100 up to a 1,000 of such impacts per season6,8,9. Additionally, other populations, such as military men and women, have registered more than 300,000 head injuries since 2001, which has manifested into the recent diagnosis of CTE within a retired military veteran10. This diagnosis parallels with 110 postmortem CTE brains of American football players and four postmortem soccer players to present a burgeoning public health issue11,12. In light of the staggering prevalence, head impact research must shift its gaze to incorporate sound, precise methods of analyzing the acute debt subconcussive hits are inducing in a variety of arenas.
The SSHM presented here is one that satisfies the current methodological need to safely induce common mechanical stresses placed upon neural tissue during contact sport activities. The implementation of this model allows investigators to meticulously manage ball traveling speed, the frequency of impacts, interval, ball placement to the head, as well as measurements of head impact magnitude13,14. While these factors are virtually impossible to control in the field setting, the SSHM provides an outlet for researchers to isolate the effects of subconcussive head impacts. Furthermore, through the elimination of confounding variables seen during play (e.g., effects from vigorous exercise, body damage, body temperature change, and hydration/perspiration), the SSHM provides a superior method of validating clinical observations.
The SSHM has direct parallels to head impacts seen specifically within the realm of sport. As such, the literature has already begun to show its utility and corroborate the findings of cumulative head impact burden from other investigators. For example, we have demonstrated that the burden of subconcussive head impacts significantly drive neuro-ophthalmologic dysfunction amongst soccer athletes13,15. In addition, as few as 10 subconcussive impacts have been shown to immediately perturb vestibular function which can be normalized after 24 h of resting16. In this methodological report, we describe the application of SSHM to safely study the effects of subconcussive head impacts and introduce one of our findings that repetitive subconcussive head impacts gradually increase the concentration of a neuron-derived blood biomarker, namely NF-L14. This finding not only substantiates previous results of NF-L presence due to repetitive subconcussive blows to the head17,18 but also validates that the SSHM can reproduce such findings in a controlled clinical manner.

<table border="0" cellpadding="0" cellspacing="0" style="width:1267px;" width="1267">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">JUGS Soccer Machine</td>
			<td style="width:179px;">JUGS Sports</td>
			<td style="width:705px;">http://jugssports.com/products/soccer-machine.html</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">SIM-G Triaxial Accelerometer</td>
			<td style="width:179px;">Triax Technologies</td>
			<td style="width:705px;">https://www.triaxtec.com/workersafety/wp-content/uploads/2017/08/SIM-G-User-Manual_V4-2-01.pdf</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo protocol of controlled subconcussive head impacts for the validation of field study data

Subconcussive hits pose a threat to neuronal health as they have shown to induce neuronal structural damage and functional impairment without causing outward symptomology and appear to be a key contributor to an irreversible neurodegenerative disease, chronic traumatic encephalopathy (CTE). In addition, athletes can incur more than 1,000 of these hits per season. The subconcussive soccer heading model (SSHM) is a relevant, reproducible, and leading method of isolating and examining the effects of these subconcussive head impacts. By controlling variables such as ball traveling speed, the frequency of impacts, interval, ball placement to the head, as well as by measuring head impact magnitude, the SSHM provides the scientific community with a superior avenue of investigating the acute subconcussive effects on neuronal health. In this paper, we demonstrate the utility of SSHM in studying a time-course expression of neurofilament-light polypeptide (NF-L) in plasma in a repeated measures fashion. NF-L is an axonal injury marker that has previously been shown to be elevated in boxers and football players following subconcussive head trauma. Thirty-four adult aged soccer players were recruited and randomly assigned to either a soccer heading (n = 18) or kicking (n = 16) group. The heading group executed 10 headers with soccer balls projected at a velocity of 25 mph over 10 min. The kicking group followed the same protocol with 10 kicks. Plasma samples were obtained before and at 0 h, 2 h, and 24 h after heading/kicking and assessed for NF-L expressions. The heading group showed a gradual increase in plasma NF-L expression and peaked at 24 h after the heading protocol, whereas the kicking group remained consistent across the time points. These results confirmed the NF-L data from clinical field studies, encouraging the use of SSHM to validate clinical subconcussion data.

The results represented here were interpreted from a previous article14, in which the SSHM was utilized as previously described. In this particular study, we aimed to show how the SSHM could induce changes in plasma levels of NF-L, which is an axonal injury marker that is hypothesized to filter out of the cranium and into the peripheral blood following head impacts.
SSHM and head kinematics
The present data were derived from 34 subjects who were eligible for analysis (heading group: n = 18, and kicking [control] group: n = 16). There were no significant differences in any demographic characteristic between the groups. Demographics and head impact kinematics are detailed in Table 1. Head impact kinematic data showed that the heading group experienced a median linear head acceleration of 31.8 g per head impact (IQR: 31.1-34.5 g) and a median rotational head acceleration of 3.56 krad/s2 per head impact (IQR: 2.93-4.04 krad/s2). In contrast, the kicking (control) group did not result in detectable levels of head acceleration (as expected) (Table 1).
SSHM effects on NF-L biomarker levels
The SSHM was able to produce the following outcomes. See Figure 1 for the visual representation of the results14. (i) There was a gradual increase in plasma NF-L expression, as illustrated by a statistically significant time effect for the head impact group, F(1,31) = 9.17, p = 0.0049. For example, 0.03 pg/mL of NF-L is estimated to increase every hour after 10 headers (SE = 0.001). (ii) There was no significant time effect for the kicking (control) group, F(1,31) = 1.20, p = 0.28. (iii) Follow-up paired t-tests with Bonferroni correction within the heading group revealed that a significant difference appeared at 24 h post-heading (3.68 &#177; 0.30 pg/mL) compared to pre-heading (3.12 &#177; 0.29 pg/mL, p = 0.0013; Cohen&#8217;s d = 1.898). (iv) Linear regression, adjusting for the baseline NF-L level, was used to assess the between-group difference at the 24 h post-intervention time point and distinguished that the NF-L level in the heading group was significantly higher than the kicking (control) group with an estimated mean difference of 0.66 pg/mL (SE = 0.22, p = 0.0025).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Variables</td>
			<td>Heading</td>
			<td>Kicking Control</td>
			<td>P-value</td>
		</tr>
		<tr>
			<td>n</td>
			<td>18</td>
			<td>16</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sex</td>
			<td>7M 11F</td>
			<td>6M 10F</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Age, y</td>
			<td>20.3 &#177; 1.5</td>
			<td>21.2 &#177; 1.4</td>
			<td>0.089</td>
		</tr>
		<tr>
			<td>BMI, kg/m2</td>
			<td>23.2 &#177; 2.4</td>
			<td>24.4 &#177; 3.2</td>
			<td>0.236</td>
		</tr>
		<tr>
			<td>No. of previous concussion</td>
			<td>0.78 &#177; 1.0</td>
			<td>0.63 &#177; 1.7</td>
			<td>0.753</td>
		</tr>
		<tr>
			<td>Soccer heading experience, y</td>
			<td>9.5 &#177; 3.6</td>
			<td>10.0 &#177; 4.5</td>
			<td>0.725</td>
		</tr>
		<tr>
			<td>Head impact kinematics, median (IQR), a</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PLA, g</td>
			<td>31.8 (31.1 &#8211; 34.5)</td>
			<td>- b</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PRA, krad/s2</td>
			<td>3.56 (2.93 &#8211; 4.04)</td>
			<td>- b</td>
			<td>-</td>
		</tr>
		<tr>
			<td colspan="4">Note: BMI, body mass index. IQR, interquartile range. PLA, peak linear acceleration. PRA, peak rotational acceleration. krad, kiloradian. a, Based on the sum of 10 soccer headers. b, Soccer kicking did not cause a detectable level of head acceleration.&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Demographics and impact kinematics by group.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59381/59381fig1.jpg" /> 
Figure 1: Changes in plasma NF-L levels before and after subconcussive impacts. In the heading group, NF-L was elevated at 24 h post-heading compared with pre-heading and 0 h post-heading time points, but the kicking (control) group remained static across all time points. The heading group&#8217;s NF-L level at 24 h post-heading was higher than that of the kicking (control) group. Data are presented as the mean &#177; SEM. NF-L = neurofilament light; SEM = standard error of the mean. The figure is reproduced from Wirsching et al.14 with permission from Mary Ann Liebert, Inc., New Rochelle, NY. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59381/59381fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Protocol of Controlled Subconcussive Head Impacts for the Validation of Field Study Data at JoVE.com

In Vivo Protocol of Controlled Subconcussive Head Impacts for the Validation of Field Study Data

The subconcussive soccer heading model is a safe and concise methodological approach to isolate and measure the effects of subconcussive head impacts.

Subconcussive hits pose a threat to neuronal health as they have shown to induce neuronal structural damage and functional impairment without causing ...

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Nanyang Technological University

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Behavior

6.3K Views.  Indiana University. The subconcussive soccer heading model is a safe and concise methodological approach to isolate and measure the effects of subconcussive head impacts.

Video: In Vivo Protocol of Controlled Subconcussive Head Impacts for the Validation of Field Study Data

Method for Simultaneous fMRI/EEG Data Collection during a Focused Attention Suggestion for Differential Thermal Sensation

In the present work, we demonstrate a method for concurrent collection of EEG/fMRI data. In our setup, EEG data are collected using a high-density 256-channel sensor net. The EEG amplifier itself is contained in a field isolation containment system (FICS), and MRI clock signals are synchronized with EEG data collection for subsequent MR artifact characterization and removal. We demonstrate this method first for resting state data collection. Thereafter, we demonstrate a protocol for EEG/fMRI data recording, while subjects listen to a tape asking them to visualize that their left hand is immersed in a cold-water bath and referred to, here, as the cold glove paradigm. Thermal differentials between each hand are measured throughout EEG/fMRI data collection using an MR compatible temperature sensor that we developed for this purpose. We collect cold glove EEG/fMRI data along with simultaneous differential hand temperature measurements both before and after hypnotic induction. Between pre and post sessions, single modality EEG data are collected during the hypnotic induction and depth assessment process. Our representative results demonstrate that significant changes in the EEG power spectrum can be measured during hypnotic induction, and that hand temperature changes during the cold glove paradigm can be detected rapidly using our MR compatible differential thermometry device.

We present a protocol for concurrent collection of EEG/fMRI data, and synchronized MR clock signal recording. We demonstrate this method using a unique paradigm whereby subjects receive &#x2018;cold glove&#x2019; instructions during scanning, and EEG/fMRI data are recorded along with hand temperature measurements both before and after hypnotic induction.

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Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.

The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. The purpose of this article is to describe the NNNS, provide video examples of the NNNS procedures and discuss the ways in which the exam has been used.

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Stereotaxic Microinjection of Viral Vectors Expressing Cre Recombinase to Study the Role of Target Genes in Cocaine Conditioned Place Preference

Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes of interest allows for enhanced temporally- and regionally-specific control of gene deletion, compared to existing methods. While conditional deletion can also be achieved by mating mice that express Cre recombinase under the control of specific gene promoters with mice carrying a floxed gene, stereotaxic microinjection allows for targeting of discrete brain areas at experimenter-determined time points of interest. In the context of cocaine conditioned place preference, and other cocaine behavioral paradigms such as self-administration or psychomotor sensitization that can involve withdrawal, extinction and/or reinstatement phases, this technique is particularly useful in exploring the unique contribution of target genes to these distinct phases of behavioral models of cocaine-induced plasticity. Specifically, this technique allows for selective ablation of target genes during discrete phases of a behavior to test their contribution to the behavior across time. Ultimately, this understanding allows for more targeted therapeutics that are best able to address the most potent risk factors that present themselves during each phase of addictive behavior.

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Microinjecting recombinant adenoassociated viral (rAAV) vectors expressing Cre recombinase into distinct mouse brain regions to selectively knockout genes ...

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
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The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

A Protocol for Housing Mice in an Enriched Environment

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent studies demonstrate that environmental enrichment decreases adiposity, increases energy expenditure, resists diet induced obesity, and causes cancer remission and inhibition in mice. EE typically consists of larger living space, a variety of &#8216;toys&#8217; to interact with, running wheels, and can include a number of other novel environmental changes. All of this fosters a more complex social engagement, cognitive and physical stimulations. Importantly, the toy location and type of toy is changed regularly, which encourages the mice to adapt to a frequently changing and novel environment. Many variables can be manipulated in EE to promote health effects in mice. Thus these approaches are difficult to control and must be properly managed to successfully replicate the associated phenotypes. Therefore, the goal of this video is to demonstrate how EE is properly set up and maintained to assure a complex, challenging, and controlled environment so that other researchers can easily reproduce the protective effects of EE against obesity and cancer.

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

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A Structured Rehabilitation Protocol for Improved Multifunctional Prosthetic Control: A Case Study

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As prosthetic development moves towards the goal of natural control, harnessing amputees&#8217; inherent ability to learn new motor skills may enable proficiency. This manuscript describes a structured rehabilitation protocol, which includes imitation, repetition, and reinforcement learning strategies, for improved multifunctional prosthetic control.

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Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

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Basic Methods for the Study of Reproductive Ecology of Fish in Aquaria

Captive-rearing observations are valuable for revealing aspects of fish behavior and ecology when continuous field investigations are impossible. Here, a series of basic techniques are described to enable observations of the reproductive behavior of a wild-caught gobiid fish, as a model, kept in an aquarium. The method focuses on three steps: collection, transport, and observations of reproductive ecology of a substrate spawner. Essential aspects of live fish collection and transport are (1) preventing injury to the fish, and (2) careful acclimation to the aquarium. Preventing harm through injuries such as scratches or a sudden change of water pressure is imperative when collecting live fish, as any physical damage is likely to negatively affect the survival and later behavior of the fish. Careful acclimation to aquaria decreases the incidence death and mitigates the shock of transport. Observations during captive rearing include (1) the identification of individual fish and (2) monitoring spawned eggs without negative effects to the fish or eggs, thereby enabling detailed investigation of the study species' reproductive ecology. The subcutaneous injection of a visible implant elastomer (VIE) tag is a precise method for the subsequent identification of individual fish, and it can be used with a wide size range of fish, with minimal influence on their survival and behavior. If the study species is a substrate spawner that deposits adhesive eggs, an artificial nest site constructed from polyvinyl chloride (PVC) pipe with the addition of a removable waterproof sheet will facilitate counting and monitoring the eggs, lessening the investigator's influence on the nest-holding and egg-guarding behavior of the fish. Although this basic method entails techniques that are seldom mentioned in detail in research articles, they are fundamental for undertaking experiments that require the captive rearing of a wild fish.

A series of basic methods to enable the study of the reproductive ecology of fish kept in aquaria are described. These are useful protocols for collecting fish using SCUBA, transporting live fish, and observing the reproductive behavior of wild-caught fish kept in aquaria.

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The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

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Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

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