R

Euh

JT

a

ARRAA

KTNIEF

1

ftpwhelm(hi

tb

SS4

i(

4

h0

Brain Research Bulletin 115 (2015) 17–22

Contents lists available at ScienceDirect

Brain Research Bulletin

j ourna l h o mepa ge: www.elsev ier .com/ locate /bra inresbul l

esearch report

nvironmental enrichment aides in functional recovery followingnilateral controlled cortical impact of the forelimb sensorimotor areaowever intranasal administration of nerve growth factor does not

ennica Young1, Timothy Pionk1, Ivy Hiatt1, Katalin Geeck1, Jeffrey S. Smith ∗

he Brain Research Laboratory, Saginaw Valley State University, University Center, MI, USA

r t i c l e i n f o

rticle history:eceived 5 March 2015eceived in revised form 2 April 2015ccepted 8 April 2015vailable online 15 April 2015

a b s t r a c t

Purpose: An injury to the forelimb sensorimotor cortex results in the impairment of motor function inanimals. Recent research has suggested that intranasal administration of nerve growth factor (NGF), aprotein naturally found in the brain, and placement into enriched environments (EE) improves motor andcognitive function after traumatic brain injury (TBI). The purpose of this study was to determine whetherNGF, EE, or the combination of both was beneficial in the recovery of motor function following TBI.

eywords:raumatic brain injuryGF

ntranasal administrationnriched environment

Results: Uninjured animals had fewer foot faults than injured animals, displaying a lesion effect. Injuredanimals housed in EE were shown to have fewer foot faults whether or not they received NGF. Injuredanimals also displayed an increased reliance on the non-impaired limb further validating a lesion effect.Conclusion: EE is an effective treatment on the recovery of motor function after a TBI. Intranasal admin-istration of NGF was found to not be an effective treatment for functional motor recovery after a TBI.

L-SMC CCI

. Introduction

Traumatic brain injury (TBI) is a common occurrence with veryew promising treatments available that could help return patientso their normal level of functioning (Greve and Zink, 2009). Therimary injury is the result of tissue damage at the site of traumaithin the brain which physically disrupts cell membranes andomeostasis leading to neuronal swelling and other neurotoxicvents while the secondary events expands the effect of the injuryeading to further physiological insults (Moppett, 2007). Within

inutes the consequences of the secondary injury can turn lethalMoppett, 2007). The secondary insults may include re-perfusion,ypoxia, edema, and ischemia all of which will contribute to the

ndividual’s outcome (Lv et al., 2013; Moppett, 2007).

A controlled cortical impact (CCI) device creates a lesion on

he animal’s brain, simulating what a TBI would be in a humanrain. The forelimb sensorimotor cortex (FL-SMC) is located on

∗ Corresponding author at: The Malcolm and Lois Field Endowed Chair in Healthcience, Department of Health Sciences, Crystal M. Lange College of Health & Humanervices, Saginaw Valley State University, 7400 Bay Road, University Center, MI8710, USA.

E-mail addresses: jennicay@siu.edu (J. Young), tdpionk@svsu.edu (T. Pionk),jhiatt@svsu.edu (I. Hiatt), kjgeeck@svsu.edu (K. Geeck), jsmith12@svsu.eduJ.S. Smith).

1 Address: Saginaw Valley State University, 7400 Bay Road, University Center, MI8710, USA.

ttp://dx.doi.org/10.1016/j.brainresbull.2015.04.003361-9230/© 2015 Elsevier Inc. All rights reserved.

© 2015 Elsevier Inc. All rights reserved.

both the left and right lobes of the brain and assists in motorfunction (Bury and Jones, 2002). A unilateral CCI to the FL-SMCwill result in one side of the animal’s body being impaired (con-tralateral) while the other side is relatively normal (ipsilateral)(Bury and Jones, 2002). Damage unilaterally to the FL-SMC cancause deficits in the somatosensory function and in the use ofthe contralateral forelimbs (Schallert et al., 2000). With this typeof injury, animals depend more on the ipsilateral side becausethey favor the limb that is still working correctly (Bury and Jones,2002). Because of the frequent use of the ipsilateral side, neu-rons of the opposite motor cortex become responsive to changesin forelimb behavior and can promote changes in neural plas-ticity (Bury and Jones, 2002; Jones et al., 1999, 2012; Schallertet al., 2000). Neural plasticity is influenced by experience, andrepetition of tests can cause a positive or negative effect on theresults (Jones et al., 1999; Schallert et al., 2000; Will and Kelche,1992). A forelimb sensorimotor cortex CCI allows researchers tocompare the contralateral side to the ipsilateral side and to deter-mine if post-injury behavior modifications will make a significantimprovement. Sensorimotor differences can be quantified using thefoot fault and limb use tests that in previous studies have beenshown to be helpful in recovery of function, as well as assessingfor behavioral and pharmacological interventions (Humm et al.,1999; Kawamata et al., 1997; Lv et al., 2013; Schallert et al.,

2000).

Enriched environment housing has been shown to improve cog-nitive and motor function after a TBI (Kline et al., 2010; Peruzzaro

1 arch B

ersuf2a2eeeesetssws(ubEitbt2tc2tTEd2rn

tcetwfontTpflp2tibiirt2

iae

8 J. Young et al. / Brain Rese

t al., 2013). An enriched environment (EE) is created when theesearch animal is housed in an interactive environment withtimulating toys and a social aspect, which by changing and manip-lating the environment of the animal has been shown to improveunctional outcome after TBI (Kline et al., 2012; Van Praag et al.,000). An enriched environment is a large single cage that encour-ges specific social interactions and behaviors (Van Praag et al.,000). The enriched environment allows the animal to explore,xercise, and interact with other animals creating a stimulatingnvironment for the research animal (Bayona et al., 2005; Hickst al., 2002). EE housing encourages novel interactions with thenvironment and other social stimuli which in turn continuallytimulates the brain by releasing neural growth factors (Bayonat al., 2005; Hicks et al., 2007). Objects, such as animal friendlyoys, can be introduced to promote physical exercise and provideensory stimulation to the animals (Grealy et al., 1999). In thetandard environment (SE), the animal is housed in a standard cage,hich does not allow for exercise and social interactions. A previous

tudy found that housing animals in SE caused memory impairmentKovesdi et al., 2011). Enriched environment also helps to stim-late organization and increase neural structures in the animal’srain (Held et al., 1985; Kovesdi et al., 2011). In previous studiesE has contributed to a better outcome of TBI by positively affect-ng neurogenesis (Kovesdi et al., 2011). Research has also shownhat animals housed in an interactive environment have increasedrain weights, thicker cortical areas, and a greater number of func-ional connections per neuron (Bayona et al., 2005; Kovesdi et al.,011). The EE increases the amount of sensory stimuli available tohe research animal and improves the plasticity of neural networksontributing to synaptogenesis (Bayona et al., 2005; Jones et al.,012; Van Praag et al., 2000; Will et al., 2004). As a rehabilitationype of treatment, EE is effective and has been shown to lessen post-BI behavioral deficits (Sozda et al., 2010; Van Praag et al., 2000).arlier studies have shown that exposure to EE increases the pro-uction of nerve growth factor in the cerebellum (Angelucci et al.,009). Animals exposed to EE have also shown an increase in neu-otrophin gene expression which promotes neuronal survival andeural plasticity (Hicks et al., 2002).

Nerve growth factor (NGF) is an essential component necessaryo regenerate damaged cells caused by a TBI or other injury andan promote the growth of new cells within brain tissue (Angeluccit al., 2009; Branchi et al., 2006). NGF is a protein naturally found inhe brain that aids in neuronal proliferation and differentiation, asell as synaptogenesis, and is thought to help in brain structure and

unction (Branchi et al., 2006). NGF also plays a role in the devel-pment and maintenance of sympathetic and sensory peripheraleurons (Lv et al., 2013). Studies have shown that NGF preventshe loss of phenotype and preserves cholinergic neurons after aBI by increasing the synthesis of choline acetyltransferase andreventing atrophy of basal forebrain cholinergic neurons (BFCN)ollowing experimental injury (Garofalo and Cuello, 1994). Regu-ar levels of NGF in the brain have been shown in other studies tolay a role in neuronal survival and neuronal plasticity (Hicks et al.,002, 2007). NGF allows neurites to sprout and also helps to restorehe function of injured neurons (Bonini et al., 2000). Administer-ng NGF intranasally allows the protein to pass through the bloodrain barrier and diffuse within the brain with less difficulty than

f given intravenously (De Rosa et al., 2005; Lv et al., 2013). Givenntranasally at 50 �l per day for 6–7 days, NGF has been shown toeduce brain edema, improve cognitive function, and motor func-ion after traumatic brain injury TBI (Lv et al., 2013; Tian et al.,012).

While EE and NGF treatments separately have been shownn previous research to be effective, a combination of the twopproaches has yet to be explored. Earlier studies have shown thatxposure to EE increases the production of nerve growth factor in

ulletin 115 (2015) 17–22

the cerebellum (Angelucci et al., 2009). Research animals exposedto EE have shown an increase in neurotrophin gene expression(Hicks et al., 2002). Regular levels of NGF and other neurotropicfactors in the brain have been shown in other studies to play a rolein neuronal survival and neuronal plasticity (Hicks et al., 2002).Previous research suggests that administering NGF and housing theresearch animals in an enriched environment may increase the lev-els of NGF after a brain injury. In one study, when normal animalswere housed in EE, an increased level of NGF was found in differentparts of the brain (Hicks et al., 2002). However, TBI EE animals withattenuated cognitive deficits did not alter NGF levels (Hicks et al.,2002). A recent study that combined EE and a chronic treatment of5-HT, a receptor agonist which regulates mood and vascular func-tion found additive effects on the recovery process of TBI (Sozdaet al., 2010).

The addition of NGF plus an EE in a TBI expands on previ-ous research; these combined treatments may increase productionof nerve growth factor along with stimulating neurons and non-neuronal formation in the injured FL-SMC. In a unilateral CCI of theFL-SMC, an NGF treatment combined with EE will help the con-tralateral side to regain some function as well as stimulate neuralplasticity. The current study was performed in order to evaluateif NGF, EE, or the combination of both will be more beneficial inattenuating deficits caused by TBI.

2. Materials and methods

2.1. Subjects and standard housing

For this study 48 male Long-Evans rats (Charles River, Portage,MI) weighing approximately 300–350 g were used. Upon arrival,animals were placed in standard environment housing 1 week priorto surgery. Standard environment consisted of the animals beinghoused in a solitary cage (26.0 cm(w), 47.0 cm(d), and 20.3 cm(h),Alternative Design, Siloam Springs, Arkansas) with access to foodand water. All animals were on a 12:12 h reversed day:night cycle.Each rat was weighed and handled for 5 min every day prior toinjury. All subjects were handled and cared for under the guidelinesset by the Institutional Animal Care and Use Committee of SaginawValley State University.

2.2. Surgical procedure of FL-SMC CCI

Animals were initially anesthetized at 5% Isoflurane and main-tained for the rest of the procedure using a nose cone with 1–3%Isoflurane. Following a midline incision, animals received eithera 6.0 mm craniotomy over the sensorimotor cortex portion ofthe left cortex 0.5 mm anterior and 4.0 mm lateral to Bregma(Kuypers and Hoane, 2010) or a sham surgery. Using an electro-magnetic CCI device fitted with a 3.0 mm impactor tip, the injurywas induced with 2.0 mm compression of the cortex at a velocityof 2.7 m/s (Kuypers and Hoane, 2010). The incision was then sta-pled together and a dime size amount of antibiotic ointment wasapplied to the incision. Animals were monitored until they regainedmovement and consciousness. Animals were then reassigned toeither enriched or standard environments. Groups consisted ofSham/SE/VEH (8), Sham/EE/VEH (8), TBI/SE/VEH (8), TBI/EE/VEH(8), TBI/SE/NGF (8), and TBI/EE/NGF (8).

2.3. Enriched environments

Enriched environment housing consisted of an expansive set-

ting (116.0 cm(w), 69.0 cm(d), and 22.0 cm(h), Alternative Design,Siloam Springs, Arkansas), a large single cage that included mean-ingful species-specific social interaction and behaviors. Fourteenmanipulative objects or toys were routinely rearranged every day

arch B

a1pd

2

panoOcep2tt2

2

lHtTf3nsD(t

bitAtt2(2l2c

2

o5CItiwspehms

J. Young et al. / Brain Rese

nd changed twice a week in the enriched housing (Grealy et al.,999; Hicks et al., 2007; Sozda et al., 2010). Food and water wasrovided ad libitum and animals were kept on a 12:12 h reverseday:night cycle.

.4. Delivery of NGF

Nerve growth factor (VWR, Radnor, PA) was dissolved in phos-hate buffer solution (PBS) at a concentration of 0.1 mg/ml and wasdministered every other day for 7 days (Lv et al., 2013). Animalsot receiving NGF were intranasally administered a vehicle (VEH)f 50 �l of PBS every other day for 7 days (De Rosa et al., 2005).ne day after TBI animals were sedated at 4% Isoflurane in a smallhamber and maintained at 3% using a modified nose cone for thentire NGF administration procedure. After sedation animals werelaced supinely with heads in an upright position (De Rosa et al.,005). Using 100 �l Hamilton microsyringes, 5 �l of either solu-ion was dropped into each nostril 10 times, each time allowing forhe solution to pass before the next drop was given (De Rosa et al.,005; Lv et al., 2013).

.5. Behavioral measures

The foot fault test was used to evaluate recovery of coordinatedimb placing during movement (Hoane et al., 2004; Kuypers andoane, 2010). Animals were tested once per day on four different

rial days. This test was conducted on trial days 2, 6, 10, and 14 postBI (Kuypers and Hoane, 2010). Animals were allowed to exploreor 3 min on an elevated grid (56.5 × 54.0 cm) with openings of.0 × 3.0 cm while being videotaped. During video analysis theumber of foot faults of the left and right forelimb along with totalteps were counted (Jones et al., 2012; Kuypers and Hoane, 2010).ata are presented as [(contralateral − ipsilateral/total step) * 100]

Jones et al., 2012). Inter-rater reliability was conducted to assesshe scoring consistency between raters.

The limb use test measures the use of forelimbs for naturalehavior of the rat and for somatosensory dysfunction following

njury (Jones et al., 2012; Kuypers and Hoane, 2010). Animals wereested post TBI on days 4, 8, 12, and 15 (Kuypers and Hoane, 2010).nimals were placed in a glass chamber (20 cm diameter by 30 cm

all) and were allowed to explore for 5 min while being video-aped (Hoane et al., 2008; Kuypers and Hoane, 2010; Schallert et al.,000). During video analysis the number of times each forelimbleft, right, both) touched the glass wall was recorded (Jones et al.,012; Kuypers and Hoane, 2010). Data are presented as % [(ipsi-

ateral limb use + 1/2 bilateral use)/total forelimb use] (Jones et al.,012). Inter-rater reliability was conducted to assess the scoringonsistency between raters.

.6. Perfusion and tissue preparation

On day 16 post TBI animals were euthanized using a lethal dosef Euthansol (398 mg/kg) and then transcardially perfused with00 ml of buffered phosphate saline (PBS, IMEB INC, San Marcos,A) followed by a fixative of 500 ml of 10% buffered formalin (IMEB

NC, San Marcos, CA) solution. After fixation the brains were sec-ioned in two separating left and right hemispheres and then placedn a 70% alcohol solution. Following extraction both hemispheres

ere processed using the Tissue-Tek vacuum infiltration proces-or (IMEB Inc., San Marco, CA, USA) and were then embedded inaraffin wax using a tissue embedding console system (Miles Sci-

ntific, Fergus Falls, MN, USA). After embedding, the right and leftemisphere of the animal’s brain was sliced using a Leica RM2255icrotome into 30 �m thick sections. Using Tru-Bond 380 adhesive

lides (True Scientific, Bellingham, WA, USA), two brain sections

ulletin 115 (2015) 17–22 19

were carefully placed onto one slide. For all 48 animals, 40 slideswere made each with two brain sections.

2.7. Hematoxylin and eosin staining

Hematoxylin and Eosin (H&E) staining was performed to staincell bodies in order to asses brain volume and neuron estimates. Ina procedure previously outlined by Peruzzaro et al. (2013) slideswere put through repeated washes to remove paraffin, rehydrate,stain, dehydrate, and coverslip. To remove the paraffin, slides wereplaced in xylene (3 × 5 min), then 100% EtOH (2 × 5 min), then 95%EtOH (2 × 5 min), 70% EtOh (1 × 5 min), followed by distilled water(1 × 5 min) to rehydrate. To stain the slides hematoxylin (1 × 2 min)was used, then a brief rinse in distilled water, followed by blu-ing solution (1 × 5 s), then another distilled rinse (1 × 5 min), then70% EtOH (1 × 5 min), and finally eosin (1 × 1 min). Dehydrationoccurred in 70% EtOH (1 × 5 min), then 95% EtOH (2 × 5 min), fol-lowed by 100% EtOH (2 × 5 min) and cleared in xylene (3 × 3 min).Slides were then coverslipped and prepared for light microscopy.

2.8. Stereological analysis

After H&E staining and coverslipping, stereological analysis wasconducted to measure volume of remaining cortex and estimatecell counts within CA1, CA2, and CA3 regions of the hippocampus.Levels used for verification were 0.3 mm, 0.6 mm, 0.9 mm, 1.2 mm,and 1.4 mm. Analyses was done using Olympus BX-61 light micro-scope with assistance from the Visiopharm Deployed Stereologysoftware. Stereological neuronal counting was done to determine iflesioned animals had significantly reduced neuronal loss. The opti-cal dissector method was used to obtain neuronal counts by usingthe global probe and sampling 5% of the CA1, CA2, and CA3 regionsof the hippocampus with a 100× magnification oil lens. The opticaldissector method determined if EE conditions and NGF interven-tions reduced the extent of neuronal loss. Randomly chosen sitesin the hippocampus were viewed and counted. Total volume of theremaining cortex was assessed using the Cavalieri estimator, whichinvolved using a point grid and sampling 100% of the cortex at a 10×magnification. Volume measures of remaining cortex were usedas an indirect measure of lesion size. Moving lateral five differentdepths were traced for each rat brain. The Cavalieri estimator usedthe product of the summed areas and the distance between sectionplanes to assess volume (Allred and Jones, 2004).

2.9. Statistical analyses

Analysis of variance (ANOVAs) tests were performed using theprocedures for general linear models (SPSS 20 for Windows) withrepeated measures as necessary. The within subject factor was theday of testing for each of the behavioral tasks and the betweensubject factor was the groups assignments. For all statistical testsp < 0.05 was considered significant. Post hoc analyses were con-ducted using Fisher’s LSD.

3. Results

3.1. Foot fault test

The number of foot faults that occurred with the contralat-eral limb was analyzed using a repeated measures analysis ofvariance (RM-ANOVA) with day being treated as the repeated mea-sure. Average inter-rater reliability was calculated to be 93.93%,

meaning that there was consistency between raters. All injuredanimals showed a deficit in forelimb coordination that improvedover time compared to shams. A significant interaction was alsoshown across days (F3,126 = 19.59, p < 0.001, Fig. 1A) and across

20 J. Young et al. / Brain Research Bulletin 115 (2015) 17–22

Fig. 1. (A) Number of foot faults that occurred with the contralateral limb were ana-lyzed. A significant interaction was also shown across days (F3,126 = 19.59, p < 0.001)and across days between groups on mean number of foot faults (F15,126 = 3.99,p < 0.001). Plotted are the mean (±S.E.M.) foot faults. Regardless of groups all animalsdecreased in foot faults over trials. (B) All sham groups (*p < 0.001) made fewer footfT(a

dpgcgtTitTdcgTe

3

tt

Fig. 2. (A) The asymmetry score demonstrates that regardless of groups thereliance on the ipsilateral limb increased across trials. A significant interactionwas shown across days (F3,126 = 8.97, p < 0.001) but not across days between groups(F15,126 = 0.988, p = 0.474). Plotted are the percentage (±S.E.M.) of forelimb asym-metry use. (B) Sham/SE/VEH (*p’s < 0.05) displayed more asymmetry comparedto animals in the TBI/SE/VEH and TBI/SE/NGF groups. Sham/EE/VEH (**p’s < 0.05)

aults than animals in TBI/SE/VEH, TBI/EE/VEH, TBI/SE/NGF and TBI/EE/NGF groups.BI/EE/VEH (**p = 0.001) made fewer foot faults in the TBI/SE/NGF group. TBI/NGF/EE***p = 0.007) made fewer foot faults than animals in the TBI/NGF/SE group. Plottedre the mean (±S.E.M.) foot faults.

ays between groups on mean number of foot faults (F15,126 = 3.99, < 0.001, Fig. 1A). A significant interaction was shown betweenroups (F5,42 = 31.54, p < 0.001, Fig. 1B). Fisher’s LSD was used toompare the performance of various groups. While traversing therid animals in the Sham/SE/VEH group made fewer foot faultshan animals in the TBI/EE/VEH (p < 0.001), TBI/SE/VEH (p < 0.001),BI/EE/NGF (p < 0.001), and TBI/SE/NGF (p < 0.001) groups. Animalsn the Sham/EE/VEH group also made fewer foot faults comparedo animals in the TBI/EE/VEH (p < 0.001), TBI/SE/VEH (p < 0.001),BI/EE/NGF (p < 0.001), and TBI/SE/NGF (p < 0.001) groups. Theseata show a lesion effect. TBI/EE/VEH made fewer foot faults whenompared to TBI/SE/NGF (p = 0.001). Animals in the TBI/EE/NGFroup performed better on foot fault task than animals in theBI/SE/NGF group (p = 0.007). These data show an environmentalffect.

.2. Limb use test

An ANOVA was implemented to analyze forelimb asymme-ry with day being treated as the repeated measure. Over trials,he forelimb asymmetry score increased. A score of 50% would

displayed more asymmetry compared to animals in TBI/SE/VEH, TBI/EE/VEH,TBI/SE/NGF and TBI/EE/NGF groups. Plotted are the percentage (±S.E.M.) of forelimbasymmetries for each group.

indicate symmetrical use of both forelimbs. An average inter-rater reliability was calculated to be 91.33% meaning that therewas consistency between raters. A significant interaction wasalso shown across days (F3,126 = 8.97, p < 0.001, Fig. 2A) but notacross days between groups (F15,126 = 0.988, p = 0.474, Fig. 2A). Asignificant interaction was shown between groups (F5,42 = 4.27,p < 0.001, Fig. 2B). The asymmetry score demonstrates that regard-less of groups the reliance on the ipsilateral limb increased.Fisher’s LSD was used to compare the performance of vari-ous groups. Sham/SE/VEH displayed more asymmetry comparedto TBI/SE/VEH (p’s = 0.017). Sham/SE/VEH also displayed moreasymmetry compared to TBI/SE/NGF (p = 0.024). Both data pointsdisplay a lesion effect. The Sham/EE/VEH group displayed moreasymmetry compared to animals in the TBI/SE/VEH (p = 0.001),TBI/EE/VEH (p = 0.003), TBI/SE/NGF (p = 0.001), and TBI/EE/NGF(p = 0.018) groups, also displaying a lesion effect.

3.3. Cortical volume estimates

A one-way ANOVA was used to analyze estimates of corticalvolume between groups. When looking at differences between left

J. Young et al. / Brain Research B

Fig. 3. (A) Sham/EE/VEH and Sham/SE/VEH (*p’s < 0.05) groups had greater cor-tical volume compared to TBI/EE/NGF, TBI/SE/NGF, TBI/EE/VEH and TBI/SE/VEHgroups. Plotted are the mean (±S.E.M.) cortical volumes for each group. (B) Ani-mnag

acpFiug(TgT(

3

hlwmpit

als in Sham/EE/VEH and Sham/SE/VEH (*p’s < 0.05) groups had a greater averageeuronal count than compared to animals in TBI/EE/NGF, TBI/SE/NGF, TBI/EE/VEHnd TBI/SE/VEH groups. Plotted are the mean (±S.E.M.) neuronal counts for eachroup.

nd right hemispheres there was a significant interaction whenomparing between group measure of cortical volume (F5,42 = 5.76,

< 0.001, Fig. 3A). Data for cortical volume were analyzed usingisher’s LSD for multiple comparisons. A lesion effect was shownn cortical volume, sham animals displayed greater cortical vol-me than the animals with TBIs. Animals in the Sham/EE/VEHroup had greater cortical volume compared to the TBI/EE/NGFp < 0.001), TBI/SE/NGF (p = 0.010), TBI/EE/VEH (p = 0.018), andBI/SE/VEH (p = 0.002) groups. The Sham/SE/VEH group also hadreater cortical volume compared to the TBI/EE/NGF (p < 0.001),BI/SE/NGF (p = 0.008), TBI/EE/VEH (p = 0.015), and TBI/SE/VEHp = .002) groups.

.4. Neuronal estimates in hippocampus

Total cell estimates of the CA1, CA2, and CA3 regions in theippocampus were performed using the one-way ANOVA. When

ooking at differences between left and right hemispheres, thereas a significant interaction when comparing between group

easures of neuronal estimates in the hippocampus (F5,42 = 3.74,

= 0.007, Fig. 3B). A Fisher’s LSD was used to analyze data. Animalsn Sham/EE/VEH groups had a greater average neuronal counthan compared to animals in TBI/EE/NGF (p = 0.020), TBI/SE/NGF

ulletin 115 (2015) 17–22 21

(p = 0.008), TBI/EE/VEH (p = 0.006), and TBI/SE/VEH (p = 0.018)groups. The Sham/SE/VEH group also had a greater average neu-ronal count than compared to animals in TBI/EE/NGF (p = 0.023),TBI/SE/NGF (p = 0.009), TBI/EE/VEH (p = 0.007), and TBI/SE/VEH(p = 0.021) groups.

4. Discussion

Foot fault data indicates that there was a lesion effect in thatall sham animals made fewer foot faults than animals with TBIs.Foot fault data also indicated that EE animals performed fewer footfaults than animals housed in SE. For the foot fault test no effectof NGF was observed however there was a significant differencewhen comparing injured animals with EE/NGF to injured animalswith SE/NGF. Animals in the TBI/EE/NGF group made fewer footfaults than animals in the TBI/SE/NGF groups meaning that theremay be a benefit to the combination of treatments however sincethere was no effect of NGF overall the EE may be the reason thisgroup performed well.

Limb use data indicates that there was a lesion effect shown bythe sham animals having more asymmetry than animals with a TBI.With the limb use data there was no significance found between EEand SE groups as well as there not being an effect of NGF or the com-bination of NGF/EE or NGF/SE. The limb use data also displays theneed for a longer study. Increased reliance of the ipsilateral limbcould be attributed to the amount of trials given over the courseof the study. For instance, Jones et al. (2012) completed the limbuse task which involved 7 trial days post TBI. In comparison, Joneset al.’s data are similar to that of the limb use data up until thefourth day. On trial days 5, 6, and 7, Jones et al. (2012) saw signif-icant improvement on the limb use task. If the current study hadallowed for more trials of the limb use test, it is possible that datawould have been similar to the foot fault task in that there wasan improvement for all animals housed in enriched environments.Motivation to use the impaired limb is lacking in this behavioralmeasure. Unlike most tasks, there is no food reward for using theimpaired limb instead the limb use task tests spontaneous, non-skilled limb use (Ploughman et al., 2009). If a reward had beenoffered to the animals in order for them to use their impaired limb,it is again possible that limb use data would have compared withthe foot fault data.

While the foot fault data shows that enriched environment isbeneficial, the mean neuronal estimates and cortical volume didnot show an EE effect. Neuronal estimates and cortical volume didhowever show a lesion effect. With histological measures there wasno effect of NGF proving valuable whether animals were housed inEE or SE. Again the duration of the study may help explains theresults. Behavioral testing took place starting two days after injurywhile neuronal estimates and cortical volume was conducted onbrains fixated at 16 days post TBI.

The chosen route of administration and the duration of the studymight explain why there was no effect of NGF observed. Adminis-tered intranasally over the course of 2 weeks, NGF has been shownto reduce brain edema after 72 h and rescue some deficits caused byAlzheimer’s disease (De Rosa et al., 2005; Lv et al., 2013). NGF thatis intraventricularly administered over 2 weeks has been shownto reduce neuronal death of septal cholinergic neurons (Kromer,1987). For this study, it may have been more beneficial to adminis-ter NGF intraventricularly to better see recovery of motor functionafter FL-SMC CCI. De Rosa et al. (2005) conducted a study where NGFwas administered intranasally over a longer duration than that of

the current study. The effects of NGF, in this study, may not havebeen completely developed as compared to studies that contin-ued behavior testing for a longer period of time post treatmenttherefore, a longer study duration is needed (Tian et al., 2012).

2 arch B

bbinmiiriFtcommificmvat

5

rhrwNf

A

atmB

R

A

A

B

B

B

B

D

d

G

2 J. Young et al. / Brain Rese

The theory of vicariation which suggests that one part of therain has the ability to substitute function for another followingrain damage can be used as a viable explanation as to why behav-

oral measures showed EE results while histological measures didot (Slavin et al., 1988). In previous research the recovery of loss ofotor or sensory function due to TBI in a short amount of time

s occasionally explained by vicariation (May et al., 2013). Thiss because the theory also suggests that spatially distant corticalegions can resume the task of lost neurons therefore quickly help-ng regain the lost motor or sensory function (May et al., 2013).rom the time of the behavioral testing to perfusion it is possiblehat vicariation took place. Given the location of the injury, the adja-ent cortex was perhaps able to reorganize within the small amountf time. This may have contributed to enriched environment ani-als performing better on the behavioral tests with no EE effect inean neuronal estimates and cortical volume. In a previous study,

t was shown that 6 h in EE produces motor and cognitive bene-ts similar to continuous EE after TBI (de Witt et al., 2011). In theurrent study, motor deficits were seen which were reduced for ani-als in EE according to foot fault data. By the time of brain fixation,

icariation due to the enriched environment may have occurredccounting for an EE and NGF/EE effect occurring in the foot faultask but not in histological measures.

. Conclusion

The data presented here suggest that EE is beneficial in theecovery of motor function after a TBI. According to these dataowever, intranasal administration of NGF is not beneficial in theecovery of function after TBI. These data also suggest that a studyith a longer duration and a different route of administration ofGF may be more productive in attempting to attenuate deficits

rom TBI.

cknowledgements

Funding was provided by the Dow Student Research and Cre-tivity Institute to Jennica Young. The authors would also like tohank Evan Nudi, Jacob Dunkerson, Kim Meerschaert, Justin Jacq-

ain, Garrick Salois, Kelsey Dubbs, and Madeleine Searles of Therain Research Laboratory at Saginaw Valley State University.

eferences

llred, R.P., Jones, T.A., 2004. Unilateral ischemic sensorimotor cortical damage infemale rats: forelimb behavioral effects and dendritic structural plasticity in thecontralateral homotopic cortex. Exp. Neurol. 190, 433–445.

ngelucci, F., De Bartolo, P., Gelfo, F., Foti, F., Cutuli, D., Bossù, P., Caltagirone,C., Petrosini, L., 2009. Increased concentrations of nerve growth factor andbrain-derived neurotrophic factor in the rat cerebellum after exposure to envi-ronmental enrichment. Cerebellum 8, 499–506.

ayona, N.A., Bitensky, J., Teasell, R., 2005. Plasticity and reorganization of the unin-jured brain. Top. Stroke Rehabil. 12, 1.

onini, S., Lambiase, A., Rama, P., Caprioglio, G., Aloe, L., 2000. Topical treatment withnerve growth factor for neurotrophic keratitis. Ophthalmology 107, 1347–1351.

ranchi, I., D’Andrea, I., Fiore, M., Di Fausto, V., Aloe, L., Alleva, E., 2006. Early socialenrichment shapes social behavior and nerve growth factor and brain-derivedneurotrophic factor levels in the adult mouse brain. Biol. Psychiatry 60, 690–696.

ury, S.D., Jones, T.A., 2002. Unilateral sensorimotor cortex lesions in adult rats facil-itate motor skill learning with the unaffected forelimb and training-induceddendritic structural plasticity in the motor cortex. J. Neurosci. 22, 8597–8606.

e Rosa, R., Garcia, A.A., Braschi, C., Capsoni, S., Maffei, L., Berardi, N., Cattaneo, A.,2005. Intranasal administration of nerve growth factor (NGF) rescues recogni-tion memory deficits in AD11 anti-NGF transgenic mice. Proc. Natl. Acad. Sci. U.S. A. 102, 3811–3816.

e Witt, B.W., Ehrenberg, K.M., McAloon, R.L., Panos, A.H., Shaw, K.E., Raghavan,

P.V., Skidmore, E.R., Kline, A.E., 2011. Abbreviated environmental enrichmentenhances neurobehavioral recovery comparably to continuous exposure aftertraumatic brain injury. Neurorehabil. Neural Repair 25, 343–350.

arofalo, L., Cuello, A., 1994. Nerve growth factor and the monosialogangliosideGM1: analogous and different in vivo effects on biochemical, morphological,

ulletin 115 (2015) 17–22

and behavioral parameters of adult cortically lesioned rats. Exp. Neurol. 125,195–217.

Grealy, M.A., Johnson, D.A., Rushton, S.K., 1999. Improving cognitive function afterbrain injury: the use of exercise and virtual reality. Arch. Phys. Med. Rehabil. 80,661–667.

Greve, M.W., Zink, B.J., 2009. Pathophysiology of traumatic brain injury. Mt. Sinai J.Med., N. Y. 76, 97–104.

Held, J.M., Gordon, J., Gentile, A., 1985. Environmental influences on locomotorrecovery following cortical lesions in rats. Behav. Neurosci. 99, 678.

Hicks, A., Hewlett, K., Windle, V., Chernenko, G., Ploughman, M., Jolkkonen, J., Weiss,S., Corbett, D., 2007. Enriched environment enhances transplanted subventricu-lar zone stem cell migration and functional recovery after stroke. Neuroscience146, 31–40.

Hicks, R., Zhang, L., Atkinson, A., Stevenon, M., Veneracion, M., Seroogy, K., 2002.Environmental enrichment attenuates cognitive deficits, but does not alter neu-rotrophin gene expression in the hippocampus following lateral fluid percussionbrain injury. Neuroscience 112, 631–637.

Hoane, M.R., Becerra, G.D., Shank, J.E., Tatko, L., Pak, E.S., Smith, M., Murashov, A.K.,2004. Transplantation of neuronal and glial precursors dramatically improvessensorimotor function but not cognitive function in the traumatically injuredbrain. J. Neurotrauma 21, 163–174.

Hoane, M.R., Gilbert, D.R., Barbre, A.B., Harrison, S.A., 2008. Magnesium dietarymanipulation and recovery of function following controlled cortical damage inthe rat. Magnes. Res. 21, 29–37.

Humm, J.L., Kozlowski, D.A., Bland, S.T., James, D.C., Schallert, T., 1999. Use-dependent exaggeration of brain injury: is glutamate involved? Exp. Neurol.157, 349–358.

Jones, T.A., Chu, C.J., Grande, L.A., Gregory, A.D., 1999. Motor skills training enhanceslesion-induced structural plasticity in the motor cortex of adult rats. J. Neurosci.19, 10153–10163.

Jones, T.A., Liput, D.J., Maresh, E.L., Donlan, N., Parikh, T.J., Marlowe, D., Kozlowski,D.A., 2012. Use-dependent dendritic regrowth is limited after unilateral con-trolled cortical impact to the forelimb sensorimotor cortex. J. Neurotrauma 29,1455–1468.

Kawamata, T., Dietrich, W.D., Schallert, T., Gotts, J.E., Cocke, R.R., Benowitz, L.I.,Finklestein, S.P., 1997. Intracisternal basic fibroblast growth factor enhancesfunctional recovery and up-regulates the expression of a molecular marker ofneuronal sprouting following focal cerebral infarction. Proc. Natl. Acad. Sci. 94,8179–8184.

Kline, A.E., McAloon, R.L., Henderson, K.A., Bansal, U.K., Ganti, B.M., Ahmed, R.H.,Gibbs, R.B., Sozda, C.N., 2010. Evaluation of a combined therapeutic regimen of8-OH-DPAT and environmental enrichment after experimental traumatic braininjury. J. Neurotrauma 27, 2021–2032.

Kline, A.E., Olsen, A.S., Sozda, C.N., Hoffman, A.N., Cheng, J.P., 2012. Evaluation of acombined treatment paradigm consisting of environmental enrichment and the5-HT1A receptor agonist buspirone after experimental traumatic brain injury. J.Neurotrauma 29, 1960–1969.

Kovesdi, E., Gyorgy, A.B., Kwon, S.-K.C., Wingo, D.L., Kamnaksh, A., Long, J.B., Kasper,C.E., Agoston, D.V., 2011. The effect of enriched environment on the outcome oftraumatic brain injury; a behavioral, proteomics, and histological study. Front.Neurosci. 5, 42.

Kromer, L.F., 1987. Nerve growth factor treatment after brain injury prevents neu-ronal death. Science 235, 214–216.

Kuypers, N.J., Hoane, M.R., 2010. Pyridoxine administration improves behavioral andanatomical outcome after unilateral contusion injury in the rat. J. Neurotrauma27, 1275–1282.

Lv, Q., Fan, X., Xu, G., Liu, Q., Tian, L., Cai, X., Sun, W., Wang, X., Cai, Q., Bao, Y., 2013.Intranasal delivery of nerve growth factor attenuates aquaporins-4-inducededema following traumatic brain injury in rats. Brain Res. 1493, 80–89.

Moppett, I., 2007. Traumatic brain injury: assessment, resuscitation and early man-agement. Br. J. Anaesth. 99, 18–31.

Peruzzaro, S.T., Gallagher, J., Dunkerson, J., Fluharty, S., Mudd, D., Hoane, M.R., Smith,J.S., 2013. The impact of enriched environment and transplantation of murinecortical embryonic stem cells on recovery from controlled cortical contusioninjury. Restor. Neurol. Neurosci. 31, 431–450.

Schallert, T., Fleming, S.M., Leasure, J.L., Tillerson, J.L., Bland, S.T., 2000. CNS plas-ticity and assessment of forelimb sensorimotor outcome in unilateral ratmodels of stroke, cortical ablation, parkinsonism and spinal cord injury. Neuro-pharmacology 39, 777–787.

Slavin, M.D., Laurence, S., Stein, D.G., 1988. Another look at vicariation. In: BrainInjury and Recovery. Springer, pp. 165–179.

Sozda, C.N., Hoffman, A.N., Olsen, A.S., Cheng, J.P., Zafonte, R.D., Kline, A.E.,2010. Empirical comparison of typical and atypical environmental enrichmentparadigms on functional and histological outcome after experimental traumaticbrain injury. J. Neurotrauma 27, 1047–1057.

Tian, L., Guo, R., Yue, X., Lv, Q., Ye, X., Wang, Z., Chen, Z., Wu, B., Xu, G., Liu, X.,2012. Intranasal administration of nerve growth factor ameliorate �-amyloiddeposition after traumatic brain injury in rats. Brain Res. 1440, 47–55.

Van Praag, H., Kempermann, G., Gage, F.H., 2000. Neural consequences of environ-mental enrichment. Nat. Rev. Neurosci. 1, 191–198.

Will, B., Kelche, C., 1992. Environmental approaches to recovery of function from

brain damage: a review of animal studies (1981 to 1991). In: Recovery fromBrain Damage. Springer, pp. 79–103.

Will, B., Galani, R., Kelche, C., Rosenzweig, M.R., 2004. Recovery from brain injuryin animals: relative efficacy of environmental enrichment, physical exercise orformal training (1990–2002). Prog. Neurobiol. 72, 167–182.