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.

All experiments described in this protocol were reviewed and approved by the Uniformed Services University Animal Care and Use Committee.&#160;
1. Craniectomy and controlled cortical impact
<ol>
	<li>Preparation of the controlled cortical impact device and surgical supplies.
	<ol>
		<li>Load a 1 mL slip-tip syringe with 0.5 mL of sterile saline for wound irrigation. Attach a 25 G needle to the syringe to control irrigation.</li>
		<li>Prepare a dilute solution of CsA in DMSO to final concent...

<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. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. 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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 border="0" cellpadding="0" cellspacing="0" style="width:774px;" width="773">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">1 ml syringes</td>
			<td style="width:201px;">Becton Dickinson (BD)</td>
			<td style="width:123px;">&#160;309659</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">1.7 ml flip top test tubes</td>
			<td>Denville</td>
			<td style="width:123px;">C2170</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">10 microliter syringe</td>
			<td>Hamilton</td>
			<td style="width:123px;">7635-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">25G Precision Glide syringe needles</td>
			<td>Becton Dickinson (BD)</td>
			<td style="width:123px;">&#160;305122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">70% ethanol</td>
			<td></td>
			<td style="width:123px;"></td>
			<td>Product of choice; varies by region</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">acetaminophen oral suspension</td>
			<td>Tylenol (Children&#39;s)</td>
			<td style="width:123px;"></td>
			<td>Dilute to 1 mg/ml in water</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">anesthetic vaporizer</td>
			<td>Vetland</td>
			<td style="width:123px;">521-11-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">animal handling cloth</td>
			<td></td>
			<td style="width:123px;"></td>
			<td>Purchase from department store</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">Betadine</td>
			<td>Purdue Products</td>
			<td style="width:123px;">NDC-67618-151-32</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">compressed oxygen</td>
			<td></td>
			<td style="width:123px;"></td>
			<td>Product of choice; varies by region</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">cyclosporine A</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">30024-100mg</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">DAB staining kit</td>
			<td>Vector Laboratories</td>
			<td style="width:123px;">SK-4100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td style="width:123px;">D8418-500ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">DMEM</td>
			<td>Invitrogen (ThermoFisher)</td>
			<td style="width:123px;">A14430-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey anti-mouse IgG antibody, HRP conjugated</td>
			<td>Jackson ImmunoResearch</td>
			<td style="width:123px;">715-035-151</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">electrical tape</td>
			<td>3M Corporation</td>
			<td style="width:123px;"></td>
			<td>Purchase from department store</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">fine tweezers</td>
			<td>Fine Science Tools</td>
			<td style="width:123px;">11254-20</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">forceps</td>
			<td>Fine Science Tools</td>
			<td style="width:123px;">91106-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">glass capillary pipettes, 1 mm OD, 0.58 mm ID</td>
			<td>World Precision Instruments</td>
			<td style="width:123px;">1B100F-3</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">High Speed Rotary Micromotor Kit</td>
			<td>Foredom Electric Co.&#160;</td>
			<td style="width:123px;">K.1070 - K.107018</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:238px;">Ideal Micro Drill Burr Set Of 5</td>
			<td>Cell Point Scientific&#160;</td>
			<td style="width:123px;">60-1000</td>
			<td></td>
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			<td height="20" style="height:20px;width:238px;">Impact One Stereotaxic Impactor for CCI&#160;</td>
			<td>Leica Biosystems</td>
			<td style="width:123px;">39463920</td>
			<td></td>
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			<td height="20" style="height:20px;width:238px;">isoflurane</td>
			<td>Baxter</td>
			<td style="width:123px;">NDC-10019-360-60</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">lab bench timers</td>
			<td>Fisher Scientific</td>
			<td style="width:123px;">14-649-17</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micropipette puller</td>
			<td>MicroData Instruments, Inc.</td>
			<td style="width:123px;">PMP-102</td>
			<td>Any puller will suffice</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">Microscope cover slips</td>
			<td>Fisherbrand</td>
			<td style="width:123px;">12-545-E</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">Microscope slide mounting medium</td>
			<td></td>
			<td style="width:123px;"></td>
			<td style="width:212px;">Product of choice</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:238px;">mirror</td>
			<td></td>
			<td style="width:123px;"></td>
			<td>Purchase from department store</td>
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			<td height="20" style="height:20px;">mouse anti-human nuclear antigen antibody</td>
			<td>Millipore</td>
			<td style="width:123px;">MAB1281</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:238px;">Mouse on Mouse blocking kit</td>
			<td>Vector Laboratories</td>
			<td style="width:123px;">BMK-2202</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">needle holder hemostat</td>
			<td>Fine Science Tools</td>
			<td style="width:123px;">12002-12</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">ophthalmic ointment</td>
			<td>Falcon Pharmaceuticals</td>
			<td style="width:123px;">NDC-61314-631-36</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">ophthalmic spring scissors</td>
			<td>Fine Science Tools</td>
			<td style="width:123px;">15018-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">plastic box</td>
			<td></td>
			<td style="width:123px;"></td>
			<td>Purchase from department store</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:238px;">plastic cylinder</td>
			<td></td>
			<td style="width:123px;"></td>
			<td>Purchase from department store</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:238px;">QSI motorized syringe pump</td>
			<td>Stoelting</td>
			<td style="width:123px;">53311</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">Removable needle compression fitting</td>
			<td>Hamilton</td>
			<td style="width:123px;">55750-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">small rodent stereotaxic frame</td>
			<td>Stoelting</td>
			<td style="width:123px;">51925</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">small scissors</td>
			<td>Fine Science Tools</td>
			<td style="width:123px;">14060-09</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">StemPro Accutase</td>
			<td>Invitrogen (ThermoFisher)</td>
			<td style="width:123px;">A1110501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">Sterile alcohol prep pads</td>
			<td>Fisherbrand</td>
			<td style="width:123px;">06-669-62</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">sterile cotton swabs/Kendall Q-tips</td>
			<td>Tyco Healthcare</td>
			<td style="width:123px;">540500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">Sterile saline</td>
			<td>Hospira</td>
			<td style="width:123px;">NDC-0409-1966-07</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">Stopwatches (2)</td>
			<td>Fisher Scientific</td>
			<td style="width:123px;">06-662-56</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">Superfrost Plus Gold microscope slides</td>
			<td>Fisherbrand</td>
			<td style="width:123px;">15-188-48</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:238px;">sutures - 5.0 silk with curved needle</td>
			<td>Oasis</td>
			<td style="width:123px;">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 ...

Our protocol demonstrates a comprehensive preclinical workflow for testing the therapeutic efficacy of human-induced pluripotent stem cells after brain injury. This model of traumatic brain injury provides a high degree of versatility and reproducibility by tightly controlling the parameters of the mechanical force and therefore providing a well-defined injury. These procedures can be used to study injury and recovery processes in other brain regions and the transplantation method is amenable to use with many cell types.Cranial drilling is challenging as the technique requires manual dexterity to master the appropriate pressure, speed, depth, and smooth movements. Document all of the events and practice extensively. To perform a unilateral craniectomy, confirm a lack of response to toe pinch in the anesthetized mouse and secure the mouse in a stereotaxic frame.Apply antibiotic ophthalmic ointment to the eyes with a sterile cotton swab and iodine-based solution to the shaved scalp area. Remove the iodine with 70%ethanol and cover the animal with a fenestrated surgical drape. Make a 1.5 to two centimeter midline incision on the scalp and use sterile cotton swabs to clean the wound and to clear the fascia left of the midline at bregma.Use an impactor probe to identify the craniectomy site and set the x equals zero, y equals zero stereotaxic reference point to bregma. Adjust the probe laterally to two millimeters left of the midline. Use a fine-tip surgically safe marker to landmark a five millimeter diameter circle around the presumptive impact area.Raise and rotate the impactor out of position and complete the five millimeter circle. Briefly return the impactor into position to verify accuracy of the five millimeter circle. Then raise and rotate the impactor out of position and use a high-speed rotary micro motor kit hand tool equipped with a round tip 0.6 or 0.8 millimeter burr drill bit to make an open hole in the skull at 70 to 80%maximum speed.Apply light pressure to the skull while drilling along the five millimeter circumferential outline within this border and use forceps to lift the resulting bone flap. Drilling through suture lines may present additional difficulty as the bone is interrupted and requires additional pressure. Blood vessels may be in close association and therefore bleeding events are likely.To induce a mild controlled cortical impact injury, clean the impactor probe with a sterile alcohol prep pad and move the impactor probe back into position over the exposed cortex. Lower the probe until it touches the dura mater surface and mark this position as z equals zero. Withdraw the piston and move to z equals minus one millimeter and discharge the piston to impact the cortex.Immediately raise the piston and move the arm out of position and irrigate the cortex with a generous volume of saline and use simple interrupted stitches and 5-0 silk suture to close the scalp incision. Then deliver 10 milligrams per kilogram of Cyclosporine A by subcutaneous injection into the scruff and place the mouse in a clean pre-warmed post-operative cage with monitoring until full recovery. Roughly 24 hours after the craniectomy, place the mouse back into the stereotaxic frame.Cover the mouse with the fenestrated surgical drape. Lavage the incision site with sterile saline to clean the site and to loosen the sutures. Use a 70%ethanol soaked cotton swab to gently sterilize the incision site and use fine tweezers and ophthalmic scissors to remove the sutures.Then irrigate the surgery and craniectomy site with abundant sterile saline. In a cell culture biosafety hood, gently swirl or tap the tube of cells to ensure a homogenous cell suspension. Use a micropipette to load approximately 7.5 microliters of cells into a Hamilton syringe through the plunger end.Holding the syringe at a 120 degree angle with the plunger end facing down, insert the plunger taking care not to introduce an air bubble between the suspension and plunger tip. Attach the gasket assembly to the pipette needle and attach the needle to the syringe. Push the plunger to move the cell suspension into the pipette needle and attach the syringe to the stereotaxic syringe pump.Advance the plunger to make sure the syringe pump assembly is working properly and move the needle into the coordinates for injection. Align the needle tip to bregma and set the x and y-coordinates to zero. Move the needle tip over the craniectomy to two millimeters lateral and minus one millimeter posterior to bregma and touch the needle tip to the dura mater surface.Raise the needle slightly then make a small incision in the dura mater at the location where the needle made contact. Set the stereotaxic coordinate to z equals zero and push the plunger to confirm that the cell suspension is flowing adequately before introducing the needle into the brain. Introduce the needle into the brain to a depth of z equals minus 1.4 millimeters to place the graft at the gray matter/white matter border of the deep cortex.Start the syringe pump to infuse the cell suspension over a 15-minute period. Use a long working distance microscope to monitor cell suspension outflow irrigating the surgery site with sterile saline during the injection to maintain tissue hydration. When all of the cells have been delivered, slowly withdraw the transplantation needle and irrigate the surgery site with additional saline before closing the incision with new sutures.Then deliver post-operative care as demonstrated. For an adhesive tape removal test, first use a small razor knife to cut yellow and red pieces of electrical tape into three by five millimeter strips on a smooth glass surface. Bring the mice into the behavior testing room for at least 30 minutes prior to the testing and use a handling cloth to restrain the mouse by the scruff of the neck and back such that the mouse holds the forepaws away from its body and head.Use tweezers to place an adhesive strip on the plantar surface of each forepaw and use delicate and consistent finger pressure to secure the strips to the paws. Quickly place the mouse into a plastic cylinder and start two stopwatches when the mouse has all four paws on the plastic testing box. Then use the two stopwatches to record the latencies for left paw notice, left paw removal, right paw notice and right paw removal.In this pilot experiment, forelimb function was compared in craniectomy alone, controlled cortical impact injury and naive mice. Mice that underwent surgery exhibited transient increased latencies to notice adhesive stimuli for one to three days immediately after the surgery and transient post-operative deficits in adhesive removal from the ipsilateral forepaw. Mice that underwent controlled cortical impact, however, exhibited significant deficits in motor performance in the forepaw contralateral to the injury compared to naive mice out of post-operative day 28.Immunostaining of mouse brain sections for human nuclear antigen reveals that human cell grafts can be clearly distinguished from host tissues in SHAM injury and controlled cortical impact brains. You must observe standard laboratory safety procedures when handling cultured human cells and when handling syringe needles that come into contact with rodents or immunosuppressive drugs. Brain tissue sections can be analyzed for the expression of neuroinflammatory markers as it is valuable to know how the graft affects the host tissue injury response and vice versa.We were surprised to find subtle sex differences in post-injury behavior outcomes. We are now investigating whether sex plays a role in the neuroimmune response to brain injury. Steady hands, patience, and practice are crucial for developing a good craniectomy technique.Collateral damage from an improper craniectomy can compromise the area that you wanna target for cell transplantation.

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JoVE Journal

Developmental Biology

10.5K vistas.  Uniformed Services University. Nuestro protocolo demuestra un flujo de trabajo preclínico completo para probar la eficacia terapéutica de las células madre pluripotentes inducidas por el hombre después de una lesión cerebral. Este modelo de lesión cerebral traumática proporciona un alto grado de versatilidad y reproducibilidad al controlar estrechamente los parámetros de la fuerza mecánica y, por lo tanto, proporcionar una lesión bien definida. Estos procedimientos se pueden utilizar para estudiar procesos de les...

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