Combining enriched environment and induced pluripotent stem … · 2019. 2. 22. · ad libitum for 24 hours a day for the duration of the study. All methods were approved by the Saginaw

Restorative Neurology and Neuroscience 32 (2014) 675–687DOI 10.3233/RNN-140408IOS Press

675

Combining enriched environment andinduced pluripotent stem cell therapy resultsin improved cognitive and motor functionfollowing traumatic brain injury

Jacob Dunkersona, Kasey E. Moritza, Jennica Younga, Tim Pionka, Kyle Finkb, Julien Rossignolb,Gary Dunbarb and Jeffrey S. Smitha,∗aThe Brain Research Laboratory, Saginaw Valley State University, University Center, MI, USAbField Neuroscience Institute Laboratory for Restorative Neurology, Central Michigan University,Mt. Pleasant, MI, USA

Abstract.Purpose: Despite advances towards potential clinically viable therapies there has been only limited success in improvingfunctional recovery following traumatic brain injury (TBI). In rats, exposure to an enriched environment (EE) improves learningand fosters motor skill development. Induced pluripotent stem cells (iPSC) have been shown to survive transplantation andinfluence the recovery process. The current study evaluated EE and iPSC as a polytherapy for remediating cognitive deficitsfollowing medial frontal cortex (mFC) controlled cortical impact (CCI) injury.Methods: Sixty adult male rats received a midline mFC CCI or sham injury and were randomly placed in either EE or stan-dard environment (SE). Seven days post-injury rats received bilateral transplantation of iPSCs or media. Behavioral measureswere conducted throughout the remainder of the study. Following behavioral analysis, brains were extracted and prepared forhistological analysis.Results: Open-field data revealed that combined therapy resulted in typical Sham/EE activity rearing patterns by the conclusion ofthe study. On the Vermicelli Handling task, rats with EE/iPSC polytherapy performed better than media-treated rats. Furthermore,rats treated with polytherapy performed equivalently to Sham/EE rats on the Morris water maze. Proficiency on the Rotarod wasconsistently better in EE when compared to SE counterparts. Confocal microscopy confirmed that iPSCs survived and migratedaway from the transplantation site.Conclusions: Overall, EE or iPSC therapy improved cognition and motor performance, however, full cognitive restoration wasseen only with the EE/iPSC treatment. These data suggest that EE/iPSC therapy should be explored as a potential, clinicallyrelevant, treatment for TBI.

∗Corresponding author: Jeffery S. Smith, Ph.D. The Malcolm andLois Field Endowed Chair in Health Sciences, Department of HealthSciences, Crystal M. Lange College of Health & Human Services,Saginaw Valley State University, 7400 Bay Road, University Center,MI 48710, USA. Tel.: +1 989 964 4503; Fax: +1 989 964 4925;E-mail: [email protected].

1. Introduction

Traumatic brain injury (TBI) occurs at a stagger-ing rate in the United States. In a recent reviewby the Center for Disease Control it was estimatedthat 1.7 million TBIs occur every year (Coronadoet al., 2012). Although TBI continues to be a leading

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676 J. Dunkerson et al. / Combining enriched environment and induced pluripotent stem cell therapy results

cause of death and disability, there are still limitedtreatment strategies to alleviate the long-standing func-tional losses associated with focal frontal lobe injuries(Moppett, 2007). Even with extensive clinical rehabili-tation people still suffer from long-term psychologicalimpairments, onset of socioemotional disorders, andmotor skill impairments (Barrash et al., 2011; Murphyet al., 2012; Neistadt, 1994). The leading cause of focalTBI in young adults is motor-vehicle collisions (MVC)(Coronado et al., 2012; Majdan et al., 2011).

Controlled cortical impact (CCI) contusion is ananimal model of TBI often used to research themechanisms of TBI and investigate novel therapeu-tic strategies for pre-clinical applications. This modelhas outcomes most similar to blunt-force focal lesionssuch as those inflicted by MVC (O’Connor et al.,2011). Symptoms induced by a bilateral medial frontalcortex (mFC) CCI injury include prolonged cognitivedeficits and impaired forelimb coordination (Hoffmanet al., 1994). The physiological consequences includegross reductions in grey and white matter, focal cellularnecrosis, apoptosis in the adjacent structures, followedby glial scar formation (Morales et al., 2005).

The brain’s ability to create new neural pathwaysthrough the ongoing process of neural plasticity hasbeen observed in both intact and injured brains (Allredet al., 2008). Following the cascade of degenerativeevents caused by the initial injury, the brain entersan elevated neuro-plastic state which makes it highlysusceptible to behavioral experiences and enrichedenvironment (EE) has also been shown to heightenneural plasticity in both intact brains and followingexperimental TBI (Allred et al., 2008; Bayona et al.,2005; Sozda et al., 2010). An EE is a housing conditiondesigned to provide an opportunity for social interac-tions, an open expanse for meandering and exercise,and a stimuli-rich environment. Traditional animalhousing (typically described as standard environment,SE) has been compared to social isolation, allows onlya limited space to roam, and reduced stimuli (Janssenet al., 2010; Peruzzaro et al., 2013). In addition, evi-dence supports that EE also has a positive impact onthe metabolic rate, vascular structure, and release ofneurotrophic factors in young, middle aged, and agedrats with TBI (Kolb et al., 2000; Maegele et al., 2005).

Along with the functional savings, there are exten-sive anatomical changes observed with exposureto EE (Bayona et al., 2005; Fares et al., 2013;Kolb et al., 2000; Maegele et al., 2005). Near thelesion, a reduction in glial scar formation allows for

increased synaptogenesis and reestablishment of func-tional neural pathways (Maegele et al., 2005). Inthe hippocampus, an upregulation of brain-derivedneurotrophic factor (BDNF) and nerve growth factor(NGF) leads to increased neurogenesis and sustainedlevels of long-term potentiation producing functionalgains in spatial memory reference (Fares et al., 2013;Maegele et al., 2005). Furthermore, the functional out-comes and anatomical augmentations have been foundto be enhanced by the length of stay in EE (Lee et al.,2013).

An additional method for improving recovery afterTBI that is currently undergoing pre-clinical evaluationis cell replacement therapy (Harting et al., 2008). Bothembryonic stem cells (eSCs) and induced pluripotentstem cells (iPSCs) possess the unique ability to pro-liferate and differentiate into any cell type, allowingthem to ameliorate many of the functional losses asso-ciated with primary and secondary cell death. Graftsof rat eSC pre-differentiated into neural lineages havebeen shown to develop into functional neurons withincreased axonal sprouting and integrated synaptic cir-cuitry within surrounding endogenous cells (Englundet al., 2002). A 12-month long study conducted byShear and colleagues (2004) demonstrated that mouse-derived eSCs, pre-differentiated into neural precursors(NPCs) and implanted in unilaterally prepared CCImice could survive in the host tissue and accelerate andmaintain functional recovery. Although eSC therapyshows great therapeutic potential, ethical delimmassurrounding the use of embryonic tissue and the poten-tial for host-rejection pose barriers to their clinicaladvancement.

These obstacles may be circumvented by usingiPSCs. In 2006, scientists successfully produced iPSCsby altering mouse somatic cells to return to pluripotentstages with the introduction of embryogentic growthfactors (Takahashi et al., 2006). These iPSCs canbe harvested and tailored from host tissue, whicheliminates the ethical problems associated with thederivation of eSCs. Furthermore, there is an extensiveliterature suggesting that the therapeutic potential ofiPSCs are equal to that of eSCs (Bilic et al., 2012;Stadtfeld et al., 2010; Wernig et al., 2008). In a studyby Wernig and colleagues (2008), mouse fibroblast-derived iPSCs were pre-differentiated into dopamineneurons, transplanted into adult rat models of Parkin-son’s disease, and found to integrate into the host tissueas functional neurons capable of synaptic transmission.Stabilized behavior was detected in four out of five of

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these rats, with four of these rats developing functionalintegration of the transplanted cells into the host tissue.Similarly, in an animal model of stroke, human iPSCs,derived from long-term expandable neuroepithelial-like stem (lt-NES) cells, were grafted into the striatumof ischemic rats. Subsequently, the lt-NES differenti-ated into neurons with sprouting axons that extendedinto the globus pallidus, ipsilateral to the transplan-tation site. Moreover, clinically relevant functionalmotor recovery was observed in the lt-NES-treated rats(Oki et al., 2012). Although it is clear that this typeof cell can survive, migrate, and impact the recoveryprocess (Gogel et al., 2011), other studies suggest addi-tional influence is required to help guide the fate of theiPSCs (Rezanejad et al., 2012; Stadtfeld et al., 2010).

Applying a combinational approach for attenuatingTBI deficits, such as transplanting eSCs and post-injury housing in EE, has been shown to improvefunctional recovery at a rate greater than either treat-ment alone (Hicks et al., 2007; Hoane et al., 2004;Peruzzaro et al., 2013). Hicks and colleauges (2007)induced focal ischemia and then treated the ratswith grafts of subventricalar zone stem cells (SVZ)while exposing them to EE, with the opportunity forvoluntary exercise. In the EE housed animals, theyfound that, along with an upregulation of endogenousstem cell proliferation, the exogenous cells tended tomigrate, intertwining with host stem cells near thelesion site. This may have been a result of EE-inducedup-regulation of host-released growth factors (such asNGF and BDNF) in the SVZ and in the neocortex,with these factors involved in recruiting endogenousstem cells for neurogenesis, synaptogenesis, dendriticaborization, functional re-construction of neural path-ways, and reducing scar formation near the ischemicsite. Although a majority of the transplanted SVZstem cells differentiated into glial cells, researchersobserved that dually treated animals demonstratedfunctional recovery of motor skills in the affected limb.

To better understand the impact of housing envi-ronment on stem cell activity in vivo, investigatorsexplored the effects of EE on the morphology oftransplanted striatal stem cells in a rodent modelof Huntington’s disease. Indeed, along with signifi-cantly elevated levels of BDNF in the dorsal striatum,exposure to EE increased dendritic spine density andresulted in more robust cell bodies in the striatal cellgrafts (Dobrossy et al., 2006). More recently, Peruz-zaro and colleagues (2013) studied the therapeuticeffects of EE, combined with neural eSCs, on deficits

caused by CCI injury in rats. They found that whileeach treatment (EE or eSC) improved behavioral out-comes, the greatest therapeutic impact on functionalrecovery came with administering eSC in the EEsetting. Injured rats treated with EE/eSC performedequally to Shams on all behavorial tests and, by the endof the study, outperformed rats that received eSC ther-apy alone. The transplanted eSCs were found to surviveand migrate, taking on neural and glial phenotypesnear the injury site. Collectively, these studies demon-strated that the EE-induced neuroplastic mechanismsthat act on endogenous cells may affect the migrationpatterns, cellular morphology, and functional integra-tion of transplanted eSCs, and indicated that EE maymeld well with a variety of co-therapies.

Although no investigators to date have tried acombinational approach of EE and iPSCs therapy inexperimental TBI, there is evidence to support a possi-ble synergistic interaction between the two treatments.Therefore, this project investigated the effects of com-bining bilateral iPSC transplantation with post-surgicalEE housing on the recovery of behavioral deficits fol-lowing midline mFC CCI injury. Based on previousstudies, the researchers hypothesized that injured ratsthat are placed in EE and received iPSC transplants willhave fewer functional deficiencies and greater incorpo-ration of iPSCs into the host brain as compared to theinjured animals in SE, regardless of iPSC transplant orEE-mediated recovery alone.

2. Methods

2.1. Subjects

Sixty adult male Long Evans rats (Charles River,Portage, Michigan, USA) approximately 90 days ofage weighing between 250–300 g upon arrival were thesubjects in the study. During the seven-day period priorto the contusion surgery, all rats were placed in standardenvironment (SE) conditions (26.0 cm W, × 47.6 cmD, × 20.3 cm H (Alternative Design Rat Cage, SiloamSprings, Arizona, USA). After the first surgical pro-cedure, the rats were reassigned to enriched or placedback into standard housing for the next 7 days leadingup to the iPSC transplantation surgery. During those14 days prior to behavioral testing, rats were handledfor 5 minutes a day and weighed to monitor fluctua-tions in health. The rats were exposed to a reversed12-hour dark/light cycle (8 : 00–20 : 00/20 : 00–8 : 00)


in order to conduct behavioral testing under white lightduring their active wake cycle. Regardless of housingenvironment, rats were provided food and water accessad libitum for 24 hours a day for the duration of thestudy. All methods were approved by the Saginaw Val-ley State University (SVSU) Institutional Care and UseCommittee (IACUC) prior to the study.

2.2. Surgical procedure

Rats were deeply anesthetized in an inductionchamber (10.16 cm W, × 10.16 cm, D × 30.48 cm H(Surgivet, Norwell, Maryland, USA) with a concentra-tion of 4% isoflurane vaporized in oxygen. The surgicalsite was shaved and prepped with betadine to main-tain aseptic condition. Once mounted on a stereotaxicdevice (Kopf, Tujunga, CA, USA), rats were kept atsurgical plane with a controlled supply of 2.5% to 3.0%isoflurane delivered through a nose cone. The surgicalsite was cleansed of betadine with 70% Ethanol (EtOH)and deionized (DI) water. After both pedal pinch andeye blink tests elicited no response, a midline incisionwas made exposing the skull. At this point the incisionsin the Sham rats were stapled closed and treated withNeosporin before being placed in a warm recovery cagewhile the anesthesia wore off. The skin and fascia oflesion rats were neatly retracted, exposing the craniumfor trepanation. A 6 mm burr hole was made centeredA/P +3 mm to bregma, the bone disc was removed anddiscarded, exposing the medial frontal cortex (mFC).Briefly, the blunt end impactor rod of cortical con-tusion impact device (Leica, Wetxlar, Germany) wascentered over the craniotomy, in contact with the dura.To induce a moderate CCI injury, the rod was retractedand the entire CCI device was lowered an additionalD/V −2.5 mm, in order to compress the cortical tissueto −2.5 mm. The impact velocity was set at 2.25 m/s(Peruzzaro et al., 2013). After impact, the lesion cav-ity was packed temporarily with a moist cotton pad tocontrol bleeding. The incision was closed with sur-gical staples and treated with Neosporin to preventinfection. Again, the rats were allowed to fully awakein a warmed standard cage before being placed intotheir permanent post-injury environment. Two groupswere contused and seven days later underwent bilat-eral iPSC transplantation; the first group was assignedto EE housing condition (n = 10) and the second wasassigned to SE housing (n = 10). Two additional groupsreceived contusion and seven days later received shamiPSC transplantation or Hank’s balanced salt solution

(HBSS, media); the first group was returned to EE(n = 8) and the second was returned to SE (n = 11).Finally, the last groups underwent sham injury surgeryand, seven days later, were anesthetized again to con-trol for the sham iPSC operation; the first group wasreturned to EE (n = 10) and the second was returned toSE (n = 11).

2.3. Housing environments

The EE housing environment consisted of largedrawer-style cages (Freedom Breeders, Turlock, CA,USA (Model Rodent 44 (116.0 cm W, × 69.0 cmD, × 22.0 cm H), lined with bedding. Eight gravity-fed water fixtures, protruding through the back of thedrawers provided hydration. A mesh tray, filled withrodent chow, was inserted into one side of the cage fornourishment. Each drawer was furnished with a varietyof 14 objects, such as PVC pipe, wooden blocks, rubbertoys, and miniature rat houses to stimulate exploration.For added novelty, the items were rearranged dailybetween behavioral testing procedures and replacedtwice a week. The SE condition in this experiment wasa single efficiency rodent cage (26.0 cm W, × 47.6 cmD, × 20.3 cm H (Alternative Design Rat Cage, SiloamSprings, AZ, USA) lined with the same litter as usedin the EE. The lid of the SE condition served as a foodhopper and held the water bottle. Regardless of housingcondition, all rats received the same rodent chow.

2.4. iPSC culture and adenovirus transduction

Induced pluripotent stem cells used in this exper-iment were generated from tail-tip fibroblasts (TTF)of adult Sprague-Dawley rats (Charles River, Rouen,France). The TTFs were expanded as described (Finket al., 2013). Briefly, two recombinant adenovirus vec-tors, one carrying Oct4, Sox2, and Klf4 (Ad-OKS) andthe other carrying c-Myc (Ad-Myc) were added to theTTF cultures and incubated until iPSC colonies werevisible. Colonies were hand selected and plated into 6-well plates containing mitomycin-c (Sigma-Aldrich,MO, USA) deactivated rat embryonic fibroblasts iniPSC medium for iPSC expansion. Cells were passed30–35 times, as described previously, pre-labeled withHoechst 33258 (Sigma-Aldrich, MO, USA), and re-suspended at a concentration of 20,000 cells/�L inHBSS (Sigma-Aldrich, MO, USA) for transplantation(Fink et al., 2013).


2.5. Transplantation

Seven days following surgery, injured rats were ran-domly selected to receive either iPSC or HBSS, media,transplantation. All rats were deeply anesthetized usingthe same procedures as described above. After tailpinch and pedal reflex elicited no response, Shamswere placed back into a warm cage until they werefully awake, whereas lesion rats were prepared forthe transplantation procedure. After being placed ina stereotaxic apparatus, surgical staples were removedand the initial incision was re-opened. Both skin andfascia were retracted, exposing the contusion site. Anybleeding was controlled with cotton swabs and moistcotton pads. A 10-�L Hamilton syringe was loadedwith either a suspension of iPSCs or HBSS for thetransplantation procedure. To prevent cross contamina-tion, two separate syringes were used for the procedure.The syringe was moved to two locations (A/P +1.0 mm,M/L ±1.5 mm, D/V −2.0 mm) to deposit 2.5 �L ofiPSCs or 2.5 �L of HBSS at a rate of 0.5 �L/minat each location in each hemisphere, mirrored at thesagittal midline (a total of ∼100,000 iPSCs were trans-planted in each subject that received iPSC therapy).The needle was kept in place for 2 minutes after eachtransplantation before being slowly raised (Peruzzaroet al., 2013). Once the transplantations were complete,the incision was closed, as previously described in thesurgical methods, and the rats were returned to theiroriginal housing environments.

2.6. Behavioral testing – Activity monitoring

A clear Plexiglas activity monitoring chamber (SanDiego Instruments, San Diego, CA, USA; 45.7 cmW, × 45.7 cm D, × 45.7 cm H) was used to measuretotal rears performed in 10 minutes. The total numberof rears was recorded by an infrared photo beam posi-tioned 12 cm above the bottom of the cage. Testing wasconducted on day 4, day 6 and day 27 following iPSCtransplantation.

2.7. Behavioral testing – Vermicelli handling test

Fine motor coordination was assessed with the Ver-micelli Handing Test (Tennant et al., 2010). Strands ofuncooked vermicelli pasta (1.5 cm diameter) were cutto lengths of 7 cm and, during the first 5 days of accli-mation (housed in SE) prior to the contusion surgery,the rats were given 4 strands of pasta a day to reduce a

neophobic response and develop skill handling for thebehavioral test (Tennant et al., 2010). All testing wasadministered and recorded in an SE cage that was linedwith bedding and topped with an empty lid. Rats weregiven one strand of pasta at a time only after consum-ing the entire first strand. Timing started when the firststrand of pasta was dropped into the cage and endedafter rats consumed all 4 strands of vermicelli pastaor after reaching a ceiling time of 15 minutes. Testingtook place once a day, on days 8 through 12, followingthe second surgery. Each session was recorded with ahigh-definition digital camera.

2.8. Behavioral testing – Morris water maze

The Morris water maze (MWM) was used to eval-uate both spatial learning and reference memory. Ablue circular fiberglass pool (∼160.0 cm in diame-ter; 60.0 cm deep) was filled with water and mixedwith white tempura, non-toxic paint for opaqueness.Four quadrants were marked off in the pool usingNorth, South, East, and West as reference points. Aclear, square escape platform (11.0 cm by 11.0 cm)was submerged 2.0 cm below the water centered inthe Northeast quadrant. All rats were tested twice aday for 10 consecutive days, starting on day 13 post-transplant. Each rat was placed in the pool facing thewall at one of the designated sites for that day of test-ing. Rats were given 90 seconds to locate the escapeplatform. If the rat failed to locate the escape platform,they were guided by hand. In either case, once on theplatform, rats were allotted 30 seconds to orient to theirsurroundings, before being placed in a dry heated hold-ing cage. A digital camera with wide lens and videotracking system (ANY-Maze, Stoleting, Wood Dale,IL, USA) was used to record the speed of the rat, dis-tance and path traveled, and the time spent in searchof the platform. The rats were tested twice a day, oncefrom a short distance (North or West) and once froma long distance (South or East). The start locationswere predetermined in a pseudo-random manner. Theinter-trial interval (ITI) was 15 minutes.

2.9. Behavioral testing – Rotarod

Locomotor skill acquisition was assessed using therotarod (RR) task (San Diego Instruments, San Diego,CA, USA) during the final 5 days of the study. Thetask consisted of a rotating polyurethane cylinder situ-ated approximately 1 m above a foam pad. The internal


rotating cylinder measured 7 cm in diameter and wasset to ramp up from 0 RPM to 30 RPM over a 60 sec-ond period. To begin each trial, rats were placed onthe cylinder facing opposite the way it was rotating.Each rat continued the test until it either fell off of therotating cylinder, breaking the infrared photo-beam,or reached the ceiling time of 180 seconds. All ratsperformed four trials a day with a 10 minute ITI.

2.10. Histology

All rats were sacrificed with a lethal dose ofEuthasol at twenty-eight days following iPSC trans-plantation. The rats were then transcardially perfusedwith 300 mL 0.9% buffered saline, followed by 300 mL10% buffered formalin (Smith et al., 2000). Afterdecapitation, brains were harvested and photographswere taken to compare Sham (Fig. 1A) to lesion rats(Fig. 1B). The hemispheres were dislocated into twopre-labeled cassettes and submerged in 70% EtOHovernight at 2◦C. To prepare for histological analysis,the tissue was processed in a tissue-tek vacuum infil-tration processor (IMEB Inc., San Marcos, California,USA) with a series of xylene, EtOH titers, DI washes,and fixation in paraffin wax. Each hemisphere wassituated on a cassette and embedded in paraffin for sec-tioning (Smith et al., 2000). The samples were slicedinto 30 �m thick sections on an automated microtomeand mounted on Tru-bond 380 slides (EMS, Hatfield,PN, USA).

Fig. 1. (A) An example of a sham injury in an adult male Long-Evans rat brain, approximately 4 months of age. (B) An example ofan mFC CCI lesion 35 days post-injury in an adult male Long-Evansrat brain, approximately 4 months of age.

2.11. Hematoxylin and eosin

A hematoxylin and eosin staining protocol previ-ously described by Peruzzaro and colleagues (2013)was selected to prepare the tissue for analysis. Slideswere passed through a series of washes to clear paraffin,rehydrate, stain, and dehydrate before coverslipping.Paraffin was cleared from the tissue with the sequen-tial washes: xylene (3 × 5 min), followed by 100%EtOH (2 × 5 min), then 95% EtOH (2 × 5 min), then70% EtOH (1 × 5 min), and rehydrated with DI water(1 × 5 min). To stain the nuclear tissue, slides wereimmersed in hematoxylin (1 × 2 min, IMEB Inc., SanMarcos, California, USA), rinsed in distilled water(1 × 5 sec), briefly dipped in Tacha’s bluing solution(1 × 5 sec, IMEB Inc., San Marcos, California, USA),rinsed again with distilled water (1 × 5 min), thensubmerged in 70% EtOH (1 × 5 min), and counter-stained with eosin (1 × 1 min, IMEB Inc., San Marcos,California, USA). To preserve the stain, slides wereimmediately dehydrated in 70% EtOH (1 × 5 min),then 95% EtOH (2 × 5 min), and cleared in xylene(3 × 3 min). Afterwards, slides were cover-slipped andallowed to cure for 48 hours. In preparation for lesionanalysis, five right sagittal sections were selected (at0.3 �m, 0.6 �m, 0.9 �m, 1.2 �m, and 1.4 �m ante-rior to bregma), studied under a light microscope(Olympus BX-61, Tokyo, Japan) and imaged with acamera (Olympus DP-72, Tokyo, Japan) mounted onthe microscope. The cortex and the CA1, CA2, CA3regions of the hippocampus were traced at the fivedifferent levels, using computer software (Visiopharmversion 4.4.4.0 for Microsoft, Hoersholm, Denmark).Actual remaining cortical volume was calculated bythe Cavalieri method. To quantify neuronal cells, 5% ofthe depth at each region of interest was randomly sam-pled using Optical Dissector (Visiopharm, Hoersholm,Denmark) and nuclear bodies were counted.

2.12. Immunohistochemistry

The tissue was prepared, sectioned, mounted, de-waxed, and rehydrated using the same method asdescribed above; however, to preserve the integrity ofthe pre-labeled iPSC cells, xylene was replaced withclear-rite (IMEB Inc., San Marcos, CA, USA). Forantigen retrieval, the slides were processed in DivaDecloaking solution (Biocare Medical, Concord, CA,USA) for 120 minutes at 80◦C. Slides were tem-pered with DI water and allowed to stand at room


temperature in the solution until fluorescent staining.One set of two primary antibodies were used to labelthe mounted sections. The primary antibodies used tolabel for the mature neuronal marker were rabbit anti-Neuronal Nuclei clone A60 (NeuN, 1 : 100, Millipore,Billerica, MA, USA) and the neuronal cytoskeletalmarker rabbit anti-Microtubule Associated Protein-2(MAP-2, 1 : 200, Millipore, Billerica, MA, USA). Eachprimary antibody solution was diluted with DaVinciGreen (Biocare Medical, Concord, CA, USA).

The secondary used, anti-rabbit IgG Dylight 594(1 : 200, Biocare Medical, Concord, CA, USA) co-labeling both NeuN and MAP-2, was administered ina solution dilution of Fluorescence Antibody Diluent(Biocare Medical, Concord, CA, USA).

The Intellipath FLX Autostainer (Biocare Medical,Concord, CA, USA) was used for immunohistochem-istry (IHC) labeling. Sections were initially washedwith TBS buffered solution (Biocare Medical, Con-cord, CA, USA) and after applying Rodent BlockR, primary antibody solutions, secondary antibodysolutions (Biocare Medical, Concord, CA, USA). Asolution of Rodent Block R was applied and incubatedfor 30 minutes to reduce endogenous IgG expres-sion and non-specific background. Next, the primaryantibody solution was applied and incubated for 90minutes. Then, the secondary antibody solution wasapplied and incubated for 90 minutes. Slides wereretrieved from the autostainer, pressed dry, and cover-slipped with Fluoro Care Anti-Fade mountant (BiocareMedical, Concord, CA, USA) and sealed with clearacrylic nail polish. All staining procedures were per-formed in the dark at room temperature.

2.13. Confocal microscopy

Location and identification of transplanted iPSCswas accomplished through use of a laser scan-ning confocal microscope (Olympus Fluoview FV10i,Tokyo, Japan). The sections were scanned at120 × magnification and imaged at eight total regionsof interest; three near the penumbra of the lesion; theCA1, CA2, and CA3 regions of the hippocampus; onein layer IV of the forelimb sensorimotor cortex; and,one from the olfactory tract. The depth at which the sec-tions were imaged remained constant for each regionand across all samples. Fluorescing mature neuronsand dendrites were identified with NeuN and MAP-2 and compared against the fluorescent label Hoechst33258 emitted by transplanted iPSCs.

2.14. Statistical analysis

The appropriate analysis of variance (ANOVA)tests were performed using the procedures for gen-eral linear models (SPSS 20.0 for Windows) withoptions for repeated measure where necessary. Thebetween-subject factor was group assignment andthe within-subject factor was day of testing for eachof the behavioral tasks. Least Significant Difference(LSD) post hoc analyses were conducted using the pro-cedure for pairwise-comparison of means. Statisticalsignificance of p < 0.05 was considered significant forall analyses. All data are shown as mean ± SEM.

3. Results

3.1. Activity monitoring

A repeated measure analysis of variance (RM-ANOVA) was used to detect differences in the totalnumber of rears within groups across days of testing.A trend in rears across days of testing approachedsignificance (F2,108 = 3.00, p = 0.054, Fig. 2A). TheTBI/iPSC/EE, TBI/iPSC/SE, Sham/EE, and Sham/SEreared fewer times across days of testing. Interest-ingly, the media treated groups demonstrated a gradualincrease in exploratory behavior by the final day of test-ing. To deduce if there was a significant difference inrearing behavior between the various treatments, datawere collapsed across days of testing. The total numberof recorded rears, however, did not reach significance(F5,54 = 2.16, p = 0.072, Fig. 2B).

3.2. Vermicelli handling test

The latency to consume 4 strands of vermicelli pastawas analyzed using a RM-ANOVA and all groups werefound to perform the task significantly faster by thefinal day of testing (F4,196 = 14.65, p < 0.001, Fig. 3A).The between groups interaction was also significant(F5,49 = 3.04, p = 0.018, Fig. 3B).

To identify where the variability between treatmentsemerged we compared groups with an LSD post hoctest. Regardless of environment, there was no sig-nificant effect when comparing either iPSC treatedgroup to the media treated groups or sham groups.The TBI/Media/EE and TBI/Media/SE groups, how-ever, took significantly longer than Shams to consumethe pasta strands (p’s < 0.05).


Fig. 2. (A) Mean number of rears counted at each testing inter-val during the study. Total reduction in rears over the course ofthe study was noted in both iPSC-treated and EE-housed groups,which approached significance (p = 0.054). (B) Total mean numberof rears recorded per group collapsed across the duration of the study.Although a significant difference in number of rears was not foundbetween groups, rats that received one or both treatments appearedto rear less than Sham/SE rats (p = 0.072).

3.3. Morris water maze

The latency to locate the submerged escape plat-form was analyzed using a RM-ANOVA. All groupsbecame significantly more efficient at locating theescape platform as testing continued (F9,486 = 52.39,p < 0.001, Fig. 4A). The group by day main effectwas also significant (F45,486 = 2.13, p < 0.001, Fig. 4B).Interestingly, the TBI/iPSC/EE group consistently per-formed at the same level as both Sham groups. Thewithin-subjects data were collapsed, revealing a sig-nificant main effect (F5,54 = 6.99, p < 0.001). An LSDpost hoc analysis was used to ascertain measureabledifferences between the various treatment approaches.There were no significant differences detected betweeneach monotherapy and injured controls. However, the

Fig. 3. (A) All groups were found to perform the Vermicelli Han-dling Test (VHT) in a significantly shorter amount of time acrossdays of testing. (B) Results from a post hoc analysis revealed a sig-nificant difference in mean latency to complete the VHT only whencomparing the TBI/Media/EE and TBI/Media/SE to both Shamgroups (∗p < 0.05). Although the TBI/iPSC/EE and TBI/iPSC/SEgroups tended to perform better than the TBI/Media/EE andTBI/Media/SE, the differences did not reach significance (p > 0.05).

iPSC/EE combination produced a significant treat-ment effect when compared to EE therapy (p = 0.009),iPSC therapy (p = 0.003), and no treatment (p < 0.001).Furthermore, the combinational approach restoredcognitive function to Sham levels.

3.4. Rotarod

Latency to fall off the rod was analyzed using aRM-ANOVA. Regardless of treatment, each groupwas able to remain on the rotating rod significantlylonger by the end of testing (F20,216 = 2.77, p < 0.001,Fig. 5A). Although all groups developed significantlybetter gross motor skills, only the Sham groupsimproved in a consistent manner. A RM-ANOVAdetected a significant main effect (F5,54 = 6.92,


Fig. 4. (A) Regardless of treatment, all groups learned to find theescape platform significantly faster as testing continued. (B) TheTBI/iPSC/EE group performed no differently than Sham/EE andSham/SE groups, overall, on mean latency to locate the escapeplatform (p > 0.05). Furthermore, the TBI/iPSC/EE, Sham/SE, andSham/EE all performed significantly better than TBI/iPSC/SE,TBI/Media/EE, and TBI/Media/SE groups on mean time to find theescape platform (∗p < 0.05).

p < 0.001, Fig. 5B). Once again, an LSD post hoc testwas used to identify variances between groups. Allgroups exposed to EE developed superior gross motorskills when compared with the SE housed equivalents.The Sham/EE and TBI/Media/EE both performedsignificantly better than TBI/Media/SE (p’s < 0.001),Sham/SE (p’s < 0.001), and TBI/iPSC/SE (p = 0.002and p = 0.001). The TBI/iPSC/EE polytherapygroup performed significantly better than Sham/SE(p = 0.023) and TBI/Media/SE (p = 0.012), but notsignificantly better than TBI/iPSC/SE.

3.5. Cortical volume

The mean total remaining cortical volume wasanalyzed using a between subjects ANOVA. A signifi-

Fig. 5. (A) A significant main effect was detected in mean latencyto fall from the rotating rod. (B) The only significant differenceswere between EE-housed rats and SE-housed rats. The TBI/iPSC/EEstayed on the rotating rod significantly longer than TBI/Media/SEand Sham/SE (∗p < 0.05). Both the TBI/Media/EE and Sham/EEperformed significantly better than all SE housed animals(∗∗p < 0.05).

cant main effect was observed (F5,47 = 7.26, p < 0.001,Fig. 6). An LSD post hoc analysis revealed that alllesion groups had significantly less (p’s < 0.05) corti-cal tissue remaining than either Sham groups, while nosignificant beneficial effects of EE or iPSC treatmentson this measure were found.

3.6. Hippocampus

The mean number of surviving neurons was ana-lyzed with a one-way ANOVA. A significant betweengroups main effect was found when comparing theTBI/Media/SE group to all other groups (F5,42 = 5.60,p < 0.001, Fig. 7). The Sham/EE group had sig-nificantly more cells present in the hippocampuswhen compared to TBI/Media/SE (p < 0.001) and


Fig. 6. Overall, experimental groups had significantly lower corticalvolumes compared to Shams (∗p < 0.05). Both TBI/iPSC/EE andTBI/Media/SE tended to have more remaining cortical tissue, whencompared to TBI/iPSC/SE and TBI/Media/EE, but the differenceswere not significant (p > 0.05).

the Sham/SE group (p = 0.016), but were no differ-ent compared to TBI/iPSC/EE, TBI/iPSC/SE, andTBI/Media/EE groups.

A significant increase in surviving neurons wasfound when comparing the TBI/iPSC/EE group(p = 0.001) and TBI/iPSC/SE group (p < 0.001) tothe TBI/Media/SE group. Cell survival in theTBI/Media/EE approached significance when com-pared to the TBI/Media/SE (p = 0.055). Interestingly,both TBI/iPSC/EE and TBI/iPSC/SE groups had sig-nificantly more cells when compared to the Sham/SEgroup (p = 0.049 and p = 0.01).

Fig. 7. A significant main effect for total number of neurons inthe hippocampus was present. A post hoc analysis revealed thatthe TBI/iPSC/EE and Sham/EE groups had significantly moreneurons when compared to both TBI/Media/SE and Sham/SE(∗p < 0.05). Interestingly, the TBI/Media/EE expressed significantlymore neurons in the hippocampus when compared to TBI/Media/SE(∗∗p < 0.05).

3.7. Immunohistochemistry

An overall search revealed that transplanted iPSCssurvived in vivo following mFC CCI. The positiveHoechst label was identified in multiple areas of thebrain, including near the penumbra of the lesion, in theFL-SMC, and in the white matter of the olfactory tract.iPSCs fluoresced only in Hoechst (Fig. 8A) and werenot co-labeled with the mature neuronal marker NeuN(Fig. 8B-C).

Fig. 8. (A) A region of interest from the sensorimotor cortex three positive Hoechst-labeled cells, two of which express immature neuronalcharacteristics. (B) An example of NeuN and MAP-2 in the same region of interest. (C) A merged image of both channels showing theHoechst-positive cells in the host tissue that were not co-labeled with NeuN or MAP-2.


4. Discussion

The current study suggests that EE, or iPSC therapy,or the combination of both treatments has a signifi-cant effect on functional cognitive and motor outcomesmFC CCI. Also, the combination of EE and iPSCtherapy, post-injury, restored functional cognitive per-formance on the MWM equal to that of Sham/EE andSham/SE groups. The EE condition, alone, fosteredsignificantly better gross motor skills on the RR task. Inaddition, histological analysis revealed that iPSC ther-apy, in either post-injury environment, was associatedwith a neuroprotective response in the hippocampus,whereas SE alone led to higher levels of hippocampalneuron loss. The neuroprotective response observed inTBI/iPSC/EE group may have contributed to superiorfunctional performance on the MWM task. In addition,both iPSC treated groups showed similar hippocampalvolumes and functional performance on the VHT andRR task closely related to Shams.

As the study progressed, rats placed in EE, thatreceived iPSC therapy, or the combination of both con-tinuously outperformed untreated rats and any form ofpost-injury treatment protected against secondary cellloss in the hippocampus. Interestingly, all treatmentgroups appeared to reach a performance plateau by thefinal five days of behavioral testing, whereas Sham/EErats continued to improve. This finding, that the ben-efits of EE increase over time, supports the findingsin a study conducted by Pham and colleagues (1999),where, after 12 months exposure to enriched housing,rats demonstrated significantly higher concentrationsof NGF in the hippocampus and improved acquisitionof spatial memory skills relative to SE rats. In the cur-rent study, the elevated levels of NGF associated withprolonged exposure to EE may have contributed to thepreservation of hippocampal cells seen in the enrichedgroups and this may have contributed to their superiorperformance on the final phases of the behavioral tasks.

Rats that received iPSC therapy had a significantpreservation of hippocampal cells when comparedto TBI/Media/SE (p < 0.001) and approached sig-nificance with TBI/Media/EE group (p = 0.055). Anumber of other researchers have found preservationof neural tissue with transplanted stem cells. Hoaneand colleagues (2004) transplanted eSCs in the ipsi-lateral cortex following a sensorimotor CCI procedureand found a significant reduction in lesion cavity sizein eSC treated animals compared to untreated ani-mals. Although their behavioral testing revealed a

significant improvement in fine motor coordination onthe vibrissae-forelimb placing test, animals failed toattain functional cognitive improvements on the MWM(Hoane et al., 2004). Impressively, iPSC therapy in thepresent study resulted in functional improvements inboth cognitive and motor outcomes, compared to theTBI/Media/SE group.

In spite of a strong neuroprotective response and thepresence of iPSCs near the injury site, iPSC monother-apy failed to produce significant functional cognitiveand motor outcomes. This is not a novel finding, asstem cell-driven therapy often results in increasedendogenous cell survival, with minimal behavioralimprovements (Harting et al., 2008; Oki et al., 2012;Shear et al., 2004). Shear and colleagues (2004) con-ducted a year-long study on the effects of transplantedneural progenitor cells (NPC) on functional recoveryfollowing a lateral frontoparietal cortex CCI. Theyfound surviving transplanted NPCs at 1 month, 3months, and 12 months post-implant securely inte-grated in the glial scar of the injury. Overall, NPCtreatment did not significantly impact behavioral out-comes of either the RR or MWM task (Shear et al.,2004). In the current study, iPSC survival in SE-housedrats failed to produce a significant therapeutic behav-ioral response; however, it did equate to greater cellularpreservation in the hippocampus. It has been suggestedthat transplanted stem cells do not directly improvebehavioral outcomes by integrating into the host tissue;rather, they function as neurotrophic pumps that guiderepair of damaged tissue (Harting et al., 2008). Inter-estingly, the TBI/iPSC/EE group experienced a similarneuroprotective response as the TBI/iPSC/SE groupin addition to the recovery of functional deficits. In apolytherapeutic study designed similarly to the currentinvestigation, Peruzzaro and colleagues (2013) foundthat EE combined with mouse eSC as a post-injurytreatment resulted in restoration of cognitive and motordeficits. They were able to locate the transplanted eSCco-labeled as mature neurons and activated glial cellsand the total number of surviving mature neurons in theTBI/eSC/EE group were no different than either Shamgroup. It is clear that post-injury environment has animpact on the function of transplanted stem cells andmay be required to activate the exogenous cells onceit has successfully integrated into the host tissue.

Although the combination of treatments leads togreater functional recovery than either treatment alonein the current model of TBI, the monotherapies didresult in significant preservation of hippocampal neu-


rons. Specifically, hippocampal neuron counts werepreserved in iPSC-treated rats that were housed in thestandard environment, indicating that the iPSCs, alone,may confer beneficial effects. Cells that positivelyexpressed the Hoechst label with morphological char-acteristics similar to immature neurons were locatednear the penumbra of the lesion as well as in the whitematter of the olfactory tract. This provides histologicalevidence that iPSCs do survive, migrate, and appearto differentiate into neural lineages. Since the trans-planted iPSCs were derived from rat tissue, however,further histological techniques to discern more specificmorphological characteristics were limited. In order toresolve this issue, future experiments will be designedwith iPSCs derived from mice or that contain fluores-cent knock-in genes. In addition to utilizing alternativeiPSCs, histological analysis will be conducted withultra-thick tissue sections rendered transparent by aclearing process known as SeeDB (Ke et al., 2013).This process will allow for greater analysis of cellular(both endogenous and exogenous) morphology.

A primary concern with the use of iPSCs and eSCsin vivo is the potential for uncontrolled proliferation(Miura et al., 2009). Although there was no apparenthistological evidence of aberrant proliferation patternsor the formation of tumors in the present experiment,there is a concern that iPSCs may form teratomas inlong-term studies (Miura et al., 2009). Further analysisshould be conducted on the efficacy of using iPSCs asa long-term therapeutic approach for treating neuro-logical injury.

The behavioral data from the current study stronglysupports the efficacy of combinational therapeuticapproaches, incorporating iPSC therapy with enrichedenvironments, as a viable therapeutic strategy forCCI models of TBI. However refinement of behav-ioral measures, so that more complex functions(such as emotional status/control, impulsivity, andattention) are evaluated, would greatly aid in under-standing potential efficacy and improve pre-clinicalvalidation. Additionally, our data currently adds tothe already mounting evidence that a combinationalapproach, which includes post-injury enrichment,significantly enhances therapeutic outcomes. Futureresearch should be focused on refinement of both ther-apeutic approaches (timing and components of EE, aswell as, dose response, timing, and location of trans-plant site for the cell therapy) in order to determinethe most effective therapeutic approach. In addition,it is of utmost importance that histological techniques

be refined to allow for quantification of transplantedcells fate including migration, differentiation, and inte-gration and to elucidate the mechanisms of how theenvironment that an organism is placed in effects therecovery process.

Acknowledgments

This study was generously funded through a contractwith the Field Neuroscience Institute to Jeffrey Smith.The authors would like to thank Madeleine Searlesand Evan Nudi for their countless contributions fortechnical support throughout the duration of the study.

References

Allred, R.P., & Jones, T.A. (2008). Experience – a double edgedsword for restorative neural plasticity after brain damage.Future Neurol, 3(2), 189-198. doi: 10.2217/14796708.3.2.189

Barrash, J., Asp, E., Markon, K., Manzel, K., Anderson, S.W., &Tranel, D. (2011). Dimensions of personality disturbance afterfocal brain damage: Investigation with the iowa scales of per-sonality change. J Clin Exp Neuropsychol, 33(8), 833-852. doi:10.1080/13803395.2011.561300

Bayona, N.A., Bitensky, J., & Teasell, R. (2005). Plasticity and reor-ganization of the uninjured brain. Top Stroke Rehabil, 12(3),1-10.

Bilic, J., & Izpisua Belmonte, J.C. (2012). Concise review: Inducedpluripotent stem cells versus embryonic stem cells: Closeenough or yet too far apart? Stem Cells, 30(1), 33-41. doi:10.1002/stem.700

Coronado, V.G., McGuire, L.C., Sarmiento, K., Bell, J., Lionbarger,M.R., Jones, C.D., et al. (2012). Trends in traumatic brain injuryin the u.S. And the public health response: 1995-2009. J SafetyRes, 43(4), 299-307. doi: 10.1016/j.jsr.2012.08.011

Dobrossy, M.D., & Dunnett, S.B. (2006). Morphological and cel-lular changes within embryonic striatal grafts associated withenriched environment and involuntary exercise. Eur J Neurosci,24(11), 3223-3233.

Englund, U., Bjorklund, A., Wictorin, K., Lindvall, O., & Kokaia, M.(2002). Grafted neural stem cells develop into functional pyra-midal neurons and integrate into host cortical circuitry. ProcNatl Acad Sci U S A, 99(26), 17089-17094.

Fares, R.P., Belmeguenai, A., Sanchez, P.E., Kouchi, H.Y., Boden-nec, J., Morales, A., et al. (2013). Standardized environmentalenrichment supports enhanced brain plasticity in healthy ratsand prevents cognitive impairment in epileptic rats. Plos One,8(1), e53888. doi: 10.1371/journal.pone.0053888

Fink, K.D., Rossignol, J., Lu, M., Leveque, X., Hulse, T.D., Crane,A.T., et al. (2013). Survival and differentiation of adenovirus-generated induced pluripotent stem cells transplanted into therat striatum. Cell Transplant. doi: 10.3727/096368913X670958


Gogel, S., Gubernator, M., & Minger, S.L. (2011). Progress andprospects: Stem cells and neurological diseases. Gene Ther,18(1), 1-6. doi: 10.1038/gt.2010.130

Harting, M.T., Baumgartner, J.E., Worth, L.L., Ewing-Cobbs, L.,Gee, A.P., Day, M.-C., & Cox, C.S., Jr. (2008). Cell therapiesfor traumatic brain injury. Neurosurg Focus, 24(3-4), E18. doi:10.3171/foc/2008/24/3-4/e17

Hicks, A.U., Hewlett, K., Windle, V., Chernenko, G., Plough-man, M., Jolkkonen, J., et al. (2007). Enriched environmentenhances transplanted subventricular zone stem cell migra-tion and functional recovery after stroke. Neuroscience, 146(1),31-40.

Hoane, M.R., Becerra, G.D., Shank, J.E., Tatko, L., Pak, E.S., Smith,M., & Murashov, A.K. (2004). Transplantation of neuronal andglial precursors dramatically improves sensorimotor functionbut not cognitive function in the traumatically injured brain. JNeurotrauma, 21(2), 163-174.

Hoffman, S.W., Fulop, Z., & Stein, D.G. (1994). Bilateral frontal cor-tical contusion in rats: Behavioral and anatomic consequences.J Neurotrauma, 11(4), 417-431.

Janssen, H., Bernhardt, J., Collier, J.M., Sena, E.S., McElduff, P.,Attia, J., et al. (2010). An enriched environment improves sen-sorimotor function post-ischemic stroke. Neurorehabil NeuralRepair, 24(9), 802-813. doi: 10.1177/1545968310372092

Ke, M.-T., Fujimoto, S., & Imai, T. (2013). Seedb: A simpleand morphology-preserving optical clearing agent for neuronalcircuit reconstruction. Nat Neurosci, 16(8), 1154-1161. doi:10.1038/nn.3447

Kolb, B., Gibb, R., & Gorny, G. (2000). Cortical plasticity and thedevelopment of behavior after early frontal cortical injury. DevNeuropsychol, 18(3), 423-444.

Lee, M.-Y., Yu, J.H., Kim, J.Y., Seo, J.H., Park, E.S., Kim, C.H.,et al. (2013). Alteration of synaptic activity-regulating genesunderlying functional improvement by long-term exposure toan enriched environment in the adult brain. Neurorehabil Neu-ral Repair, 27(6), 561-574. doi: 10.1177/1545968313481277

Maegele, M., Lippert-Gruener, M., Ester-Bode, T., Sauerland, S.,Schafer, U., Molcanyi, M., et al. (2005). Reversal of neuromotorand cognitive dysfunction in an enriched environment com-bined with multimodal early onset stimulation after traumaticbrain injury in rats. J Neurotrauma, 22(7), 772-782.

Majdan, M., Mauritz, W., Brazinova, A., Rusnak, M., Leitgeb,J., Janciak, I., & Wilbacher, I. (2011). Severity and out-come of traumatic brain injuries (tbi) with different causes ofinjury. Brain Inj, 25(9), 797-805. doi: 10.3109/02699052.2011.581642

Miura, K., Okada, Y., Aoi, T., Okada, A., Takahashi, K., Okita,K., et al. (2009). Variation in the safety of induced pluripo-tent stem cell lines. Nat Biotechnol, 27(8), 743-745. doi:10.1038/nbt.1554

Moppett, I.K. (2007). Traumatic brain injury: Assessment, resusci-tation and early management. Br J Anaesth, 99(1), 18-31. doi:10.1093/bja/aem128

Morales, D.M., Marklund, N., Lebold, D., Thompson, H.J., Pitka-nen, A., Maxwell, W.L., et al. (2005). Experimental modelsof traumatic brain injury: Do we really need to build a bettermousetrap? Neuroscience, 136(4), 971-989.

Murphy, M.P., & Carmine, H. (2012). Long-term health implicationsof individuals with tbi: A rehabilitation perspective. Neurore-habilitation, 31(1), 85-94. doi: 10.3233/nre-2012-0777

Neistadt, M.E. (1994). The neurobiology of learning: Implicationsfor treatment of adults with brain injury. Am J Occup Ther,48(5), 421-430.

O’Connor, W.T., Smyth, A., & Gilchrist, M.D. (2011). Animal mod-els of traumatic brain injury: A critical evaluation. PharmacolTher, 130(2), 106-113. doi: 10.1016/j.pharmthera.2011.01.001

Oki, K., Tatarishvili, J., Wood, J., Koch, P., Wattananit, S., Mine,Y., et al. (2012). Human-induced pluripotent stem cells formfunctional neurons and improve recovery after grafting instroke-damaged brain. Stem Cells, 30(6), 1120-1133. doi:10.1002/stem.1104

Peruzzaro, S.T., Gallagher, J., Dunkerson, J., Fluharty, S., Mudd,D., Hoane, M. R., & Smith, J.S. (2013). The impact of enrichedenvironment and transplantation of murine cortical embryonicstem cells on recovery from controlled cortical contusion injury.Restor Neurol Neurosci, 31(4), 431-450. doi: 10.3233/rnn-120299

Pham, T.M., Ickes, B., Albeck, D., Soderstrom, S., Granholm, A.C.,& Mohammed, A.H. (1999). Changes in brain nerve growthfactor levels and nerve growth factor receptors in rats exposedto environmental enrichment for one year. Neuroscience, 94(1),279-286.

Rezanejad, H., & Matin, M.M. (2012). Induced pluripotent stemcells: Progress and future perspectives in the stem cell world.Cell Reprogram, 14(6), 459-470. doi: 10.1089/cell.2012.0039

Shear, D.A., Tate, M.C., Archer, D.R., Hoffman, S.W., Hulce, V.D.,Laplaca, M.C., & Stein, D.G. (2004). Neural progenitor celltransplants promote long-term functional recovery after trau-matic brain injury. Brain Res, 1026(1), 11-22.

Smith, J.S., Fulop, Z.L., Levinsohn, S.A., Darrell, R.S., & Stein, D.G.(2000). Effects of the novel nmda receptor antagonist gacycli-dine on recovery from medial frontal cortex contusion injury inrats. Neural Plast, 7(1-2), 73-91.

Sozda, C.N., Hoffman, A.N., Olsen, A.S., Cheng, J.P., Zafonte,R.D., & Kline, A.E. (2010). Empirical comparison of typ-ical and atypical environmental enrichment paradigms onfunctional and histological outcome after experimental trau-matic brain injury. J Neurotrauma, 27(6), 1047-1057. doi:10.1089/neu.2010.1313

Stadtfeld, M., & Hochedlinger, K. (2010). Induced pluripotency:History, mechanisms, and applications. Genes Dev, 24(20),2239-2263. doi: 10.1101/gad.1963910

Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stemcells from mouse embryonic and adult fibroblast cultures bydefined factors. Cell, 126(4), 663-676.

Tennant, K.A., Asay, A.L., Allred, R.P., Ozburn, A.R., Kleim, J.A., &Jones, T.A. (2010). The vermicelli and capellini handling tests:Simple quantitative measures of dexterous forepaw function inrats and mice. J Vis Exp (41). doi: 10.3791/2076

Wernig, M., Zhao, J.-P., Pruszak, J., Hedlund, E., Fu, D., Soldner, F.,et al. (2008). Neurons derived from reprogrammed fibroblastsfunctionally integrate into the fetal brain and improve symp-toms of rats with parkinson’s disease. Proc Natl Acad Sci U S A105(15), 5856-5861. doi: 10.1073/pnas.0801677105

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