This work was supported by a grant from the Center for Neuroscience and Regenerative Medicine (CNRM, grant number G170244014). We appreciate the assistance of Mahima Dewan and Clara Selbrede in adhesive removal pilot studies. Kryslaine Radomski performed preliminary brain injury and cell transplantation surgeries. Amanda Fu and Laura Tucker of the USU CNRM Preclinical Studies core laboratory provided valuable advice on animal surgeries and behavior testing, respectively.

<ol>
	<li>Maas, A. I. R., 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=Traumatic+brain+injury:+integrated+approaches+to+improve+prevention,+clinical+care,+and+research.">Traumatic brain injury: integrated approaches to improve prevention, clinical care, and research.</a> The Lancet Neurology. 16, 987-1048 (2017).</li><li>Murray, C. 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=Disability-adjusted+life+years+(DALYs)+for+291+diseases+and+injuries+in+21+regions,+1990-2010:+a+systematic+analysis+for+the+Global+Burden+of+Disease+Study+2010.">Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010.</a> The Lancet. 380, 2197-2223 (2012).</li><li>Taylor, C. A., Bell, J. M., Breiding, M. J., Xu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+Brain+Injury-Related+Emergency+Department+Visits,+Hospitalizations,+and+Deaths+-+United+States,+2007+and+2013.">Traumatic Brain Injury-Related Emergency Department Visits, Hospitalizations, and Deaths - United States, 2007 and 2013.</a> Morbidity and mortality weekly report: Surveillance summaries. 66, 1-16 (2017).</li><li>Fehily, B., Fitzgerald, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repeated+Mild+Traumatic+Brain+Injury:+Potential+Mechanisms+of+Damage.">Repeated Mild Traumatic Brain Injury: Potential Mechanisms of Damage.</a> Cell Transplantation. 26, 1131-1155 (2017).</li><li>Kulbe, J. R., Hall, E. D. <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-integration+of+canonical+traumatic+brain+injury+secondary+injury+mechanisms+with+tau+pathology.">Chronic traumatic encephalopathy-integration of canonical traumatic brain injury secondary injury mechanisms with tau pathology.</a> Progress in Neurobiology. 158, 15-44 (2017).</li><li>Romine, J., Gao, X., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+cortical+impact+model+for+traumatic+brain+injury.">Controlled cortical impact model for traumatic brain injury.</a> Journal of Visualized Experiments. , e51781 (2014).</li><li>Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke,+cortical+ablation,+parkinsonism+and+spinal+cord+injury.">CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.</a> Neuropharmacology. 39, 777-787 (2000).</li><li>Mishra, A. 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=Decreased+resting+functional+connectivity+after+traumatic+brain+injury+in+the+rat.">Decreased resting functional connectivity after traumatic brain injury in the rat.</a> PloS ONE. 9, 95280 (2014).</li><li>Sours, 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=Default+mode+network+interference+in+mild+traumatic+brain+injury+-+a+pilot+resting+state+study.">Default mode network interference in mild traumatic brain injury - a pilot resting state study.</a> Brain Research. 1537, 201-215 (2013).</li><li>Radomski, K. L., Zhou, Q., Yi, K. J., Doughty, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+contusion+injury+disrupts+olfactory+bulb+neurogenesis+in+adult+mice.">Cortical contusion injury disrupts olfactory bulb neurogenesis in adult mice.</a> BMC Neuroscience. 14, 142 (2013).</li><li>Wang, X., Gao, X., Michalski, S., Zhao, S., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+Brain+Injury+Severity+Affects+Neurogenesis+in+Adult+Mouse+Hippocampus.">Traumatic Brain Injury Severity Affects Neurogenesis in Adult Mouse Hippocampus.</a> Journal of Neurotrauma. 33, 721-733 (2016).</li><li>Aertker, B. M., Bedi, S., Cox, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+CNS+repair+following+TBI.">Strategies for CNS repair following TBI.</a> Experimental Neurology. 275 (3), 411-426 (2016).</li><li>Kikuchi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPS+cell-derived+dopaminergic+neurons+function+in+a+primate+Parkinson's+disease+model.">Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model.</a> Nature. 548, 592-596 (2017).</li><li>Kondo, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+transplantation+of+human+iPSC-derived+glial-rich+neural+progenitors+improves+lifespan+of+ALS+mice.">Focal transplantation of human iPSC-derived glial-rich neural progenitors improves lifespan of ALS mice.</a> Stem Cell Reports. 3, 242-249 (2014).</li><li>Tong, L. 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=Inhibitory+interneuron+progenitor+transplantation+restores+normal+learning+and+memory+in+ApoE4+knock-in+mice+without+or+with+Abeta+accumulation.">Inhibitory interneuron progenitor transplantation restores normal learning and memory in ApoE4 knock-in mice without or with Abeta accumulation.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 34, 9506-9515 (2014).</li><li>Takahashi, 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=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Lischka, F. W., 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=Neonatal+mouse+cortical+but+not+isogenic+human+astrocyte+feeder+layers+enhance+the+functional+maturation+of+induced+pluripotent+stem+cell-derived+neurons+in+culture.">Neonatal mouse cortical but not isogenic human astrocyte feeder layers enhance the functional maturation of induced pluripotent stem cell-derived neurons in culture.</a> Glia. 66, 725-748 (2018).</li><li>Bouet, V., 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+adhesive+removal+test:+a+sensitive+method+to+assess+sensorimotor+deficits+in+mice.">The adhesive removal test: a sensitive method to assess sensorimotor deficits in mice.</a> Nature Protocols. 4, 1560-1564 (2009).</li><li>Fleming, S. M., Ekhator, O. R., Ghisays, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+sensorimotor+function+in+mouse+models+of+Parkinson's+disease.">Assessment of sensorimotor function in mouse models of Parkinson's disease.</a> Journal of Visualized Experiments. , e50303 (2013).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. Compact 2nd edn. , (2004).</li><li>Jacobs, G. H., Williams, G. A., Cahill, H., Nathans, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+of+novel+color+vision+in+mice+engineered+to+express+a+human+cone+photopigment.">Emergence of novel color vision in mice engineered to express a human cone photopigment.</a> Science. 315, 1723-1725 (2007).</li><li>Lundell, T. G., Zhou, Q., Doughty, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenin1+expression+in+cell+lineages+of+the+cerebellar+cortex+in+embryonic+and+postnatal+mice.">Neurogenin1 expression in cell lineages of the cerebellar cortex in embryonic and postnatal mice.</a> Developmental Dynamics: An Official Publication of the American Association of Anatomists. 238, 3310-3325 (2009).</li><li>Kempermann, G., Kuhn, H. G., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+hippocampal+neurons+in+adult+mice+living+in+an+enriched+environment.">More hippocampal neurons in adult mice living in an enriched environment.</a> Nature. 386, 493-495 (1997).</li><li>Piltti, 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=Transplantation+dose+alters+the+dynamics+of+human+neural+stem+cell+engraftment,+proliferation+and+migration+after+spinal+cord+injury.">Transplantation dose alters the dynamics of human neural stem cell engraftment, proliferation and migration after spinal cord injury.</a> Stem Cell Research. 15, 341-353 (2015).</li><li>Yu, 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=Severity+of+controlled+cortical+impact+traumatic+brain+injury+in+rats+and+mice+dictates+degree+of+behavioral+deficits.">Severity of controlled cortical impact traumatic brain injury in rats and mice dictates degree of behavioral deficits.</a> Brain Research. 1287, 157-163 (2009).</li><li>Kabadi, S. V., Hilton, G. D., Stoica, B. A., Zapple, D. N., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluid-percussion-induced+traumatic+brain+injury+model+in+rats.">Fluid-percussion-induced traumatic brain injury model in rats.</a> Nature Protocols. 5, 1552-1563 (2010).</li><li>Namjoshi, D. R., 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=Defining+the+biomechanical+and+biological+threshold+of+murine+mild+traumatic+brain+injury+using+CHIMERA+(Closed+Head+Impact+Model+of+Engineered+Rotational+Acceleration).">Defining the biomechanical and biological threshold of murine mild traumatic brain injury using CHIMERA (Closed Head Impact Model of Engineered Rotational Acceleration).</a> Experimental Neurology. 292, 80-91 (2017).</li><li>Shetty, A. K., Mishra, V., Kodali, M., Hattiangady, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+brain+barrier+dysfunction+and+delayed+neurological+deficits+in+mild+traumatic+brain+injury+induced+by+blast+shock+waves.">Blood brain barrier dysfunction and delayed neurological deficits in mild traumatic brain injury induced by blast shock waves.</a> Frontiers in Cellular Neuroscience. 8, 232 (2014).</li><li>Petraglia, A. L., Dashnaw, M. L., Turner, R. C., Bailes, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+mild+traumatic+brain+injury:+translation+of+physiological+and+anatomic+injury.">Models of mild traumatic brain injury: translation of physiological and anatomic injury.</a> Neurosurgery. 75, 34-49 (2014).</li><li>Siebold, L., Obenaus, A., Goyal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Criteria+to+define+mild,+moderate,+and+severe+traumatic+brain+injury+in+the+mouse+controlled+cortical+impact+model.">Criteria to define mild, moderate, and severe traumatic brain injury in the mouse controlled cortical impact model.</a> Experimental Neurology. 310, 48-57 (2018).</li><li>Tucker, L. B., Fu, A. H., McCabe, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+Male+and+Female+C57BL/6J+Mice+on+Motor+and+Cognitive+Tasks+Commonly+Used+in+Pre-Clinical+Traumatic+Brain+Injury+Research.">Performance of Male and Female C57BL/6J Mice on Motor and Cognitive Tasks Commonly Used in Pre-Clinical Traumatic Brain Injury Research.</a> Journal of Neurotrauma. 33, 880-894 (2016).</li><li>Rose, S. C., Fischer, A. N., Heyer, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+long+is+too+long?+The+lack+of+consensus+regarding+the+post-concussion+syndrome+diagnosis.">How long is too long? The lack of consensus regarding the post-concussion syndrome diagnosis.</a> Brain Injury. 29, 798-803 (2015).</li><li>Hurst, J. L., West, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taming+anxiety+in+laboratory+mice.">Taming anxiety in laboratory mice.</a> Nature Methods. 7, 825-826 (2010).</li><li>Li, X., 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+behavioral+testing+after+focal+ischemia+in+the+mouse:+functional+recovery+and+the+effects+of+gender.">Chronic behavioral testing after focal ischemia in the mouse: functional recovery and the effects of gender.</a> Experimental Neurology. 187, 94-104 (2004).</li><li>Andersen, A. B., Finger, S., Andersen, C. S., Hoagland, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensorimotor+cortical+lesion+effects+and+treatment+with+nimodipine.">Sensorimotor cortical lesion effects and treatment with nimodipine.</a> Physiology &amp; Behavior. 47, 1045-1052 (1990).</li><li>Al-Ali, 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=The+mTOR+Substrate+S6+Kinase+1+(S6K1)+Is+a+Negative+Regulator+of+Axon+Regeneration+and+a+Potential+Drug+Target+for+Central+Nervous+System+Injury.">The mTOR Substrate S6 Kinase 1 (S6K1) Is a Negative Regulator of Axon Regeneration and a Potential Drug Target for Central Nervous System Injury.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 37, 7079-7095 (2017).</li><li>Pleasant, 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=Rate+of+neurodegeneration+in+the+mouse+controlled+cortical+impact+model+is+influenced+by+impactor+tip+shape:+implications+for+mechanistic+and+therapeutic+studies.">Rate of neurodegeneration in the mouse controlled cortical impact model is influenced by impactor tip shape: implications for mechanistic and therapeutic studies.</a> Journal of Neurotrauma. 28, 2245-2262 (2011).</li></ol>

The authors have no conflicts of interest to disclose.

Mild CCI as a model system for testing experimental regenerative therapy 
The CCI model is a valuable tool for investigating mechanisms of tissue dysfunction after mechanical injury to the cortex. The tunability of the injury parameters is an attractive feature of this model. Altering the Z depth of impact, the velocity, or dwell time can increase or decrease severity of the injury as desired by the investigator10,25. The mild CCI model of c...

Traumatic brain injury (TBI) is a general term for the acquired injury to the brain due to either indirect mechanical forces (rotational acceleration/deceleration or contra-coup) from blows to the head or direct damage from objects or blast waves. TBI has been estimated to be the cause of roughly 9% of worldwide deaths and observed in an estimated 50 million cases per year1,2. A 2017 report from the Centers for Disease Control and Prevention estimated that in 2013, there were a total of 2.8 million hospital visits and deaths due to TBI in the United States3. Many milder TBIs go unreported every year. Serious TBI can lead to lifelong impairment of cognition, motor function, and overall quality of life. The consequences of mild TBI, especially repetitive sport-related TBI, have been only recently appreciated for their insidious health effects4,5. &#160;
Preclinical modeling is a vital component of developing new mechanistic insights and potential restorative therapy for TBI. The controlled cortical impact (CCI) model of TBI is an open-head model of mechanical contusion injury to the cortex. The impact parameters can be modified to produce CCI injuries that range from mild to severe6. CCI injuries are focal rather than diffuse, as seen with other closed head models of TBI. CCI can be performed to induce a unilateral injury, such that the contralateral cortex can serve as an internal comparator. This protocol demonstrates the characteristics of a mild CCI to a portion of the cortex that encompasses primary somatosensory and motor regions. This cortical area was chosen for its involvement in sensorimotor behaviors for which numerous behavior tests can detect injury-induced deficits7. Behavioral improvements due to therapeutic interventions for TBI can be detected, as well.
A hallmark of TBI is widespread neural dysfunction in the injured region. Injured neurons undergo cell death, and neuronal network connectivity is disrupted8,9. TBI disrupts recruitment of endogenous stem cells, which leads to further downstream behavior deficits10,11.&#160; Transplantation of neural stem cells and stem cell-derived cells has been explored as a possibility to restore function in the injured brain. In addition to the potential to restore damaged neural circuitry, transplanted cells exert paracrine effects that promote neuronal survival and functional recovery from TBI12. A variety of cell types have been transplanted preclinically to evaluate their restorative potential in models of neurologic disorders13,14,15. The recent popularization of induced pluripotent stem cell technology16 has facilitated the development of numerous human stem cell lines for experimental use. Preclinical testing with hiPSC-derived cells is an important first step to characterizing a given cell line&#8217;s potential therapeutic efficacy against human diseases. This laboratory has developed protocols for differentiating hiPSCs to neural phenotypes17 in pursuit of transplantable cells to aid recovery from traumatic brain injury.
Experiments in this protocol use a unilateral CCI to induce TBI to the left somatosensory and motor cortex of adult mice. A mild CCI injury results in a sustained functional deficit in the right forepaw that is used to track the effects of hiPSC-derived neural cell engraftment on functional recovery. Forepaw sensorimotor testing in this protocol was adapted from the methodology established by Bouet and colleagues18 and demonstrated previously by Fleming and colleagues19.&#160; This protocol outlines a complete workflow for performing an experimental brain injury, therapeutic transplantation of hiPS cells, and behavioral and histologic analysis of experimental outcome measures.

<table><tbody><tr><td>1 ml syringes</td><td>Becton Dickinson (BD)</td><td>&amp;nbsp;309659</td><td></td></tr><tr><td>1.7 ml flip top test tubes</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>10 microliter syringe</td><td>Hamilton</td><td>7635-01</td><td></td></tr><tr><td>25G Precision Glide syringe needles</td><td>Becton Dickinson (BD)</td><td>&amp;nbsp;305122</td><td></td></tr><tr><td>70% ethanol</td><td></td><td></td><td>Product of choice; varies by region</td></tr><tr><td>acetaminophen oral suspension</td><td>Tylenol (Children&#039;s)</td><td></td><td>Dilute to 1 mg/ml in water</td></tr><tr><td>anesthetic vaporizer</td><td>Vetland</td><td>521-11-22</td><td></td></tr><tr><td>animal handling cloth</td><td></td><td></td><td>Purchase from department store</td></tr><tr><td>Betadine</td><td>Purdue Products</td><td>NDC-67618-151-32</td><td></td></tr><tr><td>compressed oxygen</td><td></td><td></td><td>Product of choice; varies by region</td></tr><tr><td>cyclosporine A</td><td>Sigma-Aldrich</td><td>30024-100mg</td><td></td></tr><tr><td>DAB staining kit</td><td>Vector Laboratories</td><td>SK-4100</td><td></td></tr><tr><td>dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418-500ml</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen (ThermoFisher)</td><td>A14430-01</td><td></td></tr><tr><td>donkey anti-mouse IgG antibody, HRP conjugated</td><td>Jackson ImmunoResearch</td><td>715-035-151</td><td></td></tr><tr><td>electrical tape</td><td>3M Corporation</td><td></td><td>Purchase from department store</td></tr><tr><td>fine tweezers</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>forceps</td><td>Fine Science Tools</td><td>91106-12</td><td></td></tr><tr><td>glass capillary pipettes, 1 mm OD, 0.58 mm ID</td><td>World Precision Instruments</td><td>1B100F-3</td><td></td></tr><tr><td>High Speed Rotary Micromotor Kit</td><td>Foredom Electric Co.&amp;nbsp;</td><td>K.1070 - K.107018</td><td></td></tr><tr><td>Ideal Micro Drill Burr Set Of 5</td><td>Cell Point Scientific&amp;nbsp;</td><td>60-1000</td><td></td></tr><tr><td>Impact One Stereotaxic Impactor for CCI&amp;nbsp;</td><td>Leica Biosystems</td><td>39463920</td><td></td></tr><tr><td>isoflurane</td><td>Baxter</td><td>NDC-10019-360-60</td><td></td></tr><tr><td>lab bench timers</td><td>Fisher Scientific</td><td>14-649-17</td><td></td></tr><tr><td>Micropipette puller</td><td>MicroData Instruments, Inc.</td><td>PMP-102</td><td>Any puller will suffice</td></tr><tr><td>Microscope cover slips</td><td>Fisherbrand</td><td>12-545-E</td><td></td></tr><tr><td>Microscope slide mounting medium</td><td></td><td></td><td>Product of choice</td></tr><tr><td>mirror</td><td></td><td></td><td>Purchase from department store</td></tr><tr><td>mouse anti-human nuclear antigen antibody</td><td>Millipore</td><td>MAB1281</td><td></td></tr><tr><td>Mouse on Mouse blocking kit</td><td>Vector Laboratories</td><td>BMK-2202</td><td></td></tr><tr><td>needle holder hemostat</td><td>Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>ophthalmic ointment</td><td>Falcon Pharmaceuticals</td><td>NDC-61314-631-36</td><td></td></tr><tr><td>ophthalmic spring scissors</td><td>Fine Science Tools</td><td>15018-10</td><td></td></tr><tr><td>plastic box</td><td></td><td></td><td>Purchase from department store</td></tr><tr><td>plastic cylinder</td><td></td><td></td><td>Purchase from department store</td></tr><tr><td>QSI motorized syringe pump</td><td>Stoelting</td><td>53311</td><td></td></tr><tr><td>Removable needle compression fitting</td><td>Hamilton</td><td>55750-01</td><td></td></tr><tr><td>small rodent stereotaxic frame</td><td>Stoelting</td><td>51925</td><td></td></tr><tr><td>small scissors</td><td>Fine Science Tools</td><td>14060-09</td><td></td></tr><tr><td>StemPro Accutase</td><td>Invitrogen (ThermoFisher)</td><td>A1110501</td><td></td></tr><tr><td>Sterile alcohol prep pads</td><td>Fisherbrand</td><td>06-669-62</td><td></td></tr><tr><td>sterile cotton swabs/Kendall Q-tips</td><td>Tyco Healthcare</td><td>540500</td><td></td></tr><tr><td>Sterile saline</td><td>Hospira</td><td>NDC-0409-1966-07</td><td></td></tr><tr><td>Stopwatches (2)</td><td>Fisher Scientific</td><td>06-662-56</td><td></td></tr><tr><td>Superfrost Plus Gold microscope slides</td><td>Fisherbrand</td><td>15-188-48</td><td></td></tr><tr><td>sutures - 5.0 silk with curved needle</td><td>Oasis</td><td>MV-682</td><td></td></tr></tbody></table>

controlled cortical impact model of mouse brain injury with therapeutic transplantation of human induced pluripotent stem cell-derived neural cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

Craniectomy surgery facilitates experimental brain injury and therapeutic cell transplantation: the controlled cortical impact model of brain injury and subsequent cell transplantation therapy require careful removal of the overlying skull. The craniectomy may be performed on any dorsal surface of the skull to permit manipulations to the brain region of interest. The diagram in Figure 1 depicts a 5 mm diameter craniectomy schematic to uncover primary somatosensory and motor ...

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Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...

controlled-cortical-impact-model-mouse-brain-injury-with-therapeutic

Research

JoVE Journal

Developmental Biology

10.7K Views.  Uniformed Services University. Ons protocol toont een uitgebreide preklinische workflow voor het testen van de therapeutische werkzaamheid van door de mens geïnduceerde pluripotente stamcellen na hersenletsel. Dit model van traumatisch hersenletsel biedt een hoge mate van veelzijdigheid en reproduceerbaarheid door de parameters van de mechanische kracht strak te controleren en dus een goed gedefinieerd letsel te bieden. Deze procedures kunnen worden gebruikt om letsel- en herstelprocessen in andere hersengebieden te bestu...

Video: Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Medicine

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Neuroscience

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the United States. In fact, TBI is a contributing factor to over a third of all injury-related mortality. Nonetheless, the cellular and molecular mechanisms underlying the pathophysiology of TBI are poorly understood. Thus, preclinical models of TBI capable of replicating the injury mechanisms pertinent to TBI in human patients are a critical research need. The controlled cortical impact (CCI) model of TBI utilizes a mechanical device to directly impact the exposed cortex. While no model can full recapitulate the disparate injury patterns and heterogeneous nature of TBI in human patients, CCI is capable of inducing a wide range of clinically applicable TBI. Furthermore, CCI is easily standardized allowing investigators to compare results across experiments as well as across investigative groups. The following protocol is a detailed description of applying a severe CCI with a commercially available impacting device in a murine model of TBI.

Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

Transplantation of human pluripotent stem cell-derived GABAergic neurons generated by neuronal programming could be a potential treatment approach for neurodevelopmental disorders. This protocol describes the generation and transplantation of human stem cell-derived GABAergic neuronal precursors into the brains of neonatal mice, allowing long-term investigation of grafted neurons and evaluation of their therapeutic potential.

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using ...

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the lack of proper animal models that fully mimic human diseases and the poor availability of post-mortem human brain tissue. Adult human nervous tissue culture offers the possibility to study different aspects of neurological disorders. Molecular, cellular, and biochemical mechanisms could be easily addressed in this system, as well as testing and validating drugs or different treatments, such as cell-based therapies. This method combines long-term organotypic cultures of the adult human cortex, obtained from epileptic patients undergoing resective surgery, and ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors. This method will allow the study of cell survival, neuronal differentiation, the formation of synaptic inputs and outputs, and the electrophysiological properties of human-derived cells after transplantation into intact adult human cortical tissue. This approach is an important step prior to the development of a 3D human disease modeling platform that will bring basic research closer to the clinical translation of stem cell-based therapies for patients with different neurological disorders and allow the development of new tools for reconstructing damaged neural circuits.

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

This protocol describes long-term organotypic cultures of adult human cortex combined with ex vivo intracortical transplantation of induced pluripotent stem cell-derived cortical progenitors, which present a novel methodology to further test stem cell-based therapies for human neurodegenerative disorders.

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

A Human Cerebral Organoid Model of Neural Cell Transplantation

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For the central nervous system, although xenotransplantation remains state-of-the-art, such models are technically challenging, limited in throughput, and expensive. Moreover, the environmental signals present do not perfectly cross-react with human cells. This paper presents an inexpensive, accessible, and high-throughput-compatible model for the transplantation and tracking of human neural cells into human cerebral organoids. These organoids can be easily generated from human induced pluripotent stem cells using commercial kits and contain the key cell types of the cerebrum. 
We first demonstrate this transplant protocol with the injection of EGFP-labeled human iPSC-derived neural progenitor cells (NPCs) into these organoids. We next discuss considerations for tracking the growth of these cells in the organoid by live-cell fluorescence microscopy and demonstrate the tracking of transplanted EGFP-labeled NPCs in an organoid over a 4 month period. Finally, we present a protocol for the sectioning, cyclic immunofluorescent staining, and imaging of the transplanted cells in their local context. The organoid transplantation model presented here allows the long-term (at least 4 months) tracking of transplanted human cells directly in a human microenvironment with an inexpensive and simple-to-perform protocol. It, thus, represents a useful model both for neural cell therapies (transplants) and, likely, for modeling central nervous system (CNS) tumors in a more microenvironmentally accurate manner.

Here, we describe a protocol for the transplantation and tracking of labeled neural cells into human cerebral organoids.

The advancement of cell transplantation approaches requires model systems that allow an accurate assessment of transplanted cell functional potency. For ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

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