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Stem Cell Research (2012) 9, 1–8

REGULAR ARTICLE

Intravenous and intra-arterial administration of bonemarrow mononuclear cells after focal cerebralischemia: Is there a difference inbiodistribution and efficacy?

Andréia Vasconcelos-dos-Santos a, b, c,⁎, Paulo Henrique Rosado-de-Castro b, c, d,Sergio Augusto Lopes de Souza d, Juliana daCosta Silva a, Alane BernardoRamos a,Gabriel Rodriguez de Freitas a, Lea Mirian Barbosa da Fonseca d,Bianca Gutfilen d, Rosalia Mendez-Otero a, b, c

a Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazilb Programa de Terapia Celular/PROTECEL, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazilc Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem — INBEB, Rio de Janeiro, Brazild Departamento de Radiologia, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro,Rio de Janeiro, Brazil

Received 14 June 2011; received in revised form 27 January 2012; accepted 16 February 2012Available online 28 February 2012

Abstract Intravascular delivery of cells has been increasingly used in stroke models and clinical trials. We compared thebiodistribution and therapeutic effects of bone marrow mononuclear cells (BMMCs) delivered by intra-arterial (IA) or intra-venous (IV) injection after cortical ischemia. For the biodistribution analyses, BMMCs were labeled with 99mTechnetium(99mTc). At 2 h, gamma-well counting of the brain and of the other organs evaluated did not show differences betweenthe non-ischemic and ischemic groups or between injection routes, and the organs with the highest uptake were the liverand lungs, with low uptake in the brain. At 24 h, the liver maintained the highest activity, and a marked decrease wasseen in pulmonary uptake in all groups. At this time point, although the activity in the brain remained low, the lesionedhemisphere showed greater homing than the contralateral hemisphere, for both the IV and IA ischemic groups. Histologicalanalysis by CellTrace labeling indicated similar homing between both routes in the peri-infarct region 24 h after

⁎ Corresponding author at: Instituto de Biofísica Carlos Chagas Filho Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro,Av. Carlos Chagas Filho, 373 Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ CEP 21941-902, Brazil. Fax: +55 21 2280 8193.

E-mail address: andreia@biof.ufrj.br (A. Vasconcelos-dos-Santos).

1873-5061/$ - see front matter © 2012 Elsevier B.V. All rights reserved.doi:10.1016/j.scr.2012.02.002

2 A. Vasconcelos-dos-Santos et al.

transplantation and functional recovery was observed in both groups up to 11 weeks after the lesion. In conclusion, trans-plantation of BMMCs by IA or IV routes may lead to similar brain homing and therapeutic efficacy after experimental stroke.

© 2012 Elsevier B.V. All rights reserved.

Introduction

Stroke remains a leading cause of adult disability worldwide.In Brazil it is the leading cause of death, with a mortalityrate of approximately 51.6 deaths per 100,000 inhabitants(Garcia et al., 2009) and shows the highest case-fatality ratein Latin America (Lotufo and Bensenor, 2009). Recently, cell-based therapies have emerged as a promising tool for thetreatment of stroke (Baker et al., 2007; Hicks et al., 2009;Lindvall and Kokaia, 2010; Ohtaki et al., 2008; Shimada andSpees, 2011) and different cell types and routes have beenused in these studies. In order to be able to translate these re-sults to a clinical setting, more information is required regard-ing the safety and efficacy of the different cell types, doses,and routes of administration. Therapy with BMMCs has led tofunctional improvement in animal models of focal cerebral is-chemia when the cells were transplanted either intravenouslyor intra-arterially (Brenneman et al., 2010; de VasconcelosDos Santos et al., 2010; Giraldi-Guimaraes et al., 2009; Iihoshiet al., 2004; Nakano-Doi et al., 2010). Independent of the de-livery route, several studies using labeled cells from differenttissues and species donors suggest the existence of a high rateof cell entrapment in peripheral organs (Detante et al., 2009;Fischer et al., 2009; Gao et al., 2001). Moreover, the numberof injected cells reaching the brain parenchyma seems to besmall (Detante et al., 2009; Lappalainen et al., 2008). Despitethe small number of cells found in the brain tissue, functionaleffects have been observed in the cell-treated animals, sug-gesting that peripheral mechanisms may play a systemic rolein cell therapy, i.e., the cells do not necessarily need toreach the central nervous system in order to trigger their ther-apeutic effects (Borlongan et al., 2004; Mendez-Otero et al.,2007). In this respect, a careful evaluation of the biodistribu-tion of the transplanted cells and a correlation with the func-tional efficacy may provide a better understanding of themechanisms involved in the therapeutic effects observed. Nu-clear Medicine techniques provide valuable means for moni-toring cell therapies in vivo, with high image quality. Inaddition, these techniques also allow us to estimate the num-ber of cells in different organs and tissues by counting the ac-tivity in the isolated organs (Detante et al., 2009; Lassance etal., 2009; Quintanilha et al., 2008). Our working hypothesis isthat the effect of BMMCs may not depend on the intravascularmodality of administration. Therefore, the present study ex-amined the therapeutic effect after IV and IA administrationsof BMMCs. We also investigated the distribution of thesecells, to determinewhether there are differences in effective-ness between these routes of administration.

Results

Whole-body nuclear imaging

Whole-body scintigraphies performed at 2 h after transplan-tation indicated high activity in the head after IA injection, both

in ischemic (Figure 1) and non-ischemic groups (data notshown). After IV injection, no marked uptake was seen in thehead in ischemic (Figure 1) or non-ischemic groups (data notshown). High activity in the lungs, liver and kidneys was ob-served in all groups in this time frame. Scintigraphy of the isolat-ed brain, liver, spleen, kidneys, and lungs and of the headwithout the brain showed a high uptake in the head of IA animalsand low uptake in the brain (Supplemental Figure 1).

After 24 h, the activity decreased significantly due to the 6-hhalf-life of 99mTc, and the image resolution was poor. Neverthe-less, a clear reduction could be observed in the lung uptake, andactivity was limitedmainly to the liver and kidneys in all groups.Also, the activity in the head of intra-arterial groups observed at2 h was no longer present (Figure 1).

Biodistribution by isolated organ gamma-well counting

At 2 h after 99mTc-BMMC transplantation, no difference in thegamma-well counting was observed between non-ischemicand ischemic groups or between intra-arterial and intravenousinjections in the different organs evaluated (Figure 2). Thetotal activity in the organs evaluated was approximately55.2% of the injected dose and did not differ among groups.Moreover, no difference in the activity was observed betweenthe right and left hemispheres at 2 h, in both ischemic andnon-ischemic groups (Figure 2). At 24 h, only 12.3% of the initialactivity injected remained in the organs, and the only organthat showed a difference in distribution among groups wasthe brain. At this time point, the left hemisphere showedhigher activity than the right hemisphere, for both the intrave-nous (5.9±3.9×10−3%ID/g vs. 1.8±1.2×10−3% ID/g; p=0.0065;paired t-test; Figure 3) and intra-arterial groups (4.2±3.3×10−3%ID/g vs. 1.6±0.8×10−3%ID/g; p=0.0472; Figure 3).In the non-ischemic animals, no difference in activity be-tween hemispheres was seen (Figure 3). At 2 h, the organwith the highest percentage of uptake in all groups was theliver (32.5±4.5%), followed by the lungs (9.6±4.8%), kidneys(8.7±4.2%) and spleen (3.3±1.5%). With respect to the per-centage of uptake per gram of tissue, the lungs showed thehighest activity (5.7±3.4% ID/g), followed by the kidneys(3.6±2.4% ID/g), spleen (3.4±1.6% ID/g) and liver (2.9±0.6%ID/g). At 24 h, the organ with the highest percentage of up-take was the liver (8.1±1.7%), followed by the kidneys (3.0±1.3%) and spleen (0.86±0.2%). With respect to the percentageof uptake per gram of tissue, the kidneys showed the highestactivity (1.3±0.7% ID/g), followed by the spleen (1.0±0.2%ID/g) and liver (0.6±0.1% ID/g). The high activity in the lungsobserved in in vivo images and quantified in gamma-wellcounting indicated an initial trapping at 2 h and a delayed re-distribution at 24 h, with a decrease of 97% of its initial up-take, the greatest among the organs studied.

BMMCs in the periphery of the ischemic lesion

To evaluate whether the uptake in the brain in the gamma-well counting was indeed correlated with the presence of

Figure 1 Representative whole-body images demonstrating the biodistribution of BMMCs labeled with 99mTc, 2 h and 24 h (n=6 foreach group) after intravenous or intra-arterial injection in ischemic rats. The intra-arterial group had significant activity in the headat 2 h. The remaining activity could be seen in the lungs, liver, kidneys and bladder in all groups at 2 h, and in the liver and kidneys at24 h.

3Biodistribution and efficacy of bone marrow mononuclear cells after focal cerebral ischemia

transplanted cells in the ischemic hemisphere, we labeledthe BMMCs with CellTrace and performed a histologicalanalysis. A small number of labeled BMMCs were foundonly in the infarct and peri-infarct regions 24 h after trans-plantation, and no cells were found in the contralateralhemisphere. Figure 4A shows a photomicrograph demon-strating fluorescence-labeled BMMCs that migrated to thecerebral peri-infarct parenchyma. Cells could not be quan-tified in the lesion core due to the formation of the cavita-tion. There was no difference in the number of CellTrace-labeled BMMCs in ischemic animals 24 h after IV and IAtransplantations (Figure 4B).

Figure 2 Distribution of BMMCs labeled with 99mTc 2 h (A) and 24 h(n=4) and ischemic rats (n=6) by gamma-well counting. Tracer act(%dose/organ; A and B) and dose per gram of organ (%ID/g; C and D

BMMCs promote therapeutic effects independent ofthe administration route

We have previously demonstrated functional recoveryafter intravenous transplantation of BMMCs in an animalmodel of cortical ischemia (de Vasconcelos Dos Santos etal., 2010; Giraldi-Guimaraes et al., 2009). In the presentstudy, we investigated whether there are differences intherapeutic efficacy between intra-arterial and intravenousadministration routes. To answer this question, we used thecylinder test to assess functional recovery (Schallert, 2006;Schallert et al., 2001). This test is a sensitive tool to

(B) after intravenous or intra-arterial injection in non-ischemicivity is expressed as the percentage of injected dose per organ).

Figure 3 Brain distribution of BMMCs labeled with 99mTc, 2 h (A) and 24 h (B) after intravenous or intra-arterial injection innon-ischemic (n=4) and ischemic (n=6) rats by gamma-well counting. Tracer activity is expressed as the percentage of injecteddose per gram of organ (%ID/g) * pb0.05 paired t-test.

4 A. Vasconcelos-dos-Santos et al.

evaluate sensorimotor function in the thermocoagulationmodel of ischemia (de Vasconcelos Dos Santos et al., 2010;Giraldi-Guimaraes et al., 2009). We observed significantfunctional improvement after administration of BMMCs inboth groups, independent of the route of administration(Figure 5). The pre-ischemia score showed no asymmetry be-fore lesion induction, as expected, whereas, 1 day after le-sion, all ischemic animals showed clear asymmetry in theuse of the forelimbs, preferentially using the limb ipsilateralto the cortical lesion, and were therefore not affected bythe procedure (Figure 5). BMMCs were administered 1 PID,and from the 6th day after injection, evident recovery ofsensorimotor function was observed in both the IA and IVgroups. The repeated measures two-way ANOVA comparingthe treated groups with the saline group revealed significanteffects of time (F=34.91; pb0.0001), treatment (F=48.08;pb0.0001), and interaction between time and treatment(F=6780; pb0.0001). The analysis with two-way ANOVAand Bonferroni post-test showed significant differences inthe symmetry score between the treated groups (IA and IV)and the saline group at each PID starting 7 days after ische-mia (Supplemental Figure 2).

Discussion

An ever-increasing number of reports have demonstratedthat in animal subjects, BMMCs improve functional outcomeafter ischemia (Baker et al., 2007; Brenneman et al., 2010;Giraldi-Guimaraes et al., 2009; Iihoshi et al., 2004; Kamiyaet al., 2008). More recently, these cells have also been eval-uated for safety and feasibility in clinical trials in patientswith stroke (Battistella et al., 2011; Mendez-Otero et al.,2007; Mendonça et al., 2006). In these studies, the cellswere delivered intra-arterially by a catheter in the middle

cerebral artery, and the results showed that no patientsexhibited adverse effects after transplantation, or worsen-ing in neurological performance. However, IA injection re-quires a cerebral angiography, an invasive procedure thatinvolves infrequent but significant risks, including newstrokes. A retrospective study reviewing cerebral angiogra-phies in nearly 20,000 patients has indicated that the riskof complications due to the procedure is approximately 2%and that the risk of a new stroke is approximately 0.14%(Kaufmann et al., 2007). Besides the intrinsic risks of a cere-bral angiography, it is possible that the injection of cells maycreate risks such as embolisms. A preclinical study usingMSCs found that despite the benefits of intra-arterial deliv-ery of stem cells to the ischemic brain, there was a clearrisk of vascular occlusion and also a large increase in mortal-ity (67% for IA transplantation compared to 7% in non-transplanted animals) (Walczak et al., 2008). Similarly, Liet al. observed a high mortality rate (41%) after IA adminis-tration, compared with intra-cerebral (17%) and IV (8%), in-dicating that IV administration may be safer than the otherroutes (Li et al., 2010). On the other hand, a study that per-formed IA administration of BMMCs did not show alteration inthe cerebral perfusion, suggesting that this effect may be re-lated to the type of transplanted cells (Brenneman et al.,2010). Even if the IA route is shown to provide greater hom-ing in the lesion, more studies are required to elucidate ifthe IA route is more effective than the IV and of comparablesafety. This is especially relevant when considering that dif-ferent groups have suggested that the effect of cell therapyin stroke may not be directly related to the presence of cellsin the brain, since it is possible to observe functional recov-ery without the presence of cells in the cerebral parenchyma(Borlongan et al., 2004) or with few cells, as shown by stud-ies involving different types of cell therapies after IA or IVadministration (Bacigaluppi et al., 2009; Brenneman et al.,

Figure 4 BMMCs in the periphery of the injury. Fluorescencephotomicrography of the lesioned ipsilateral hemisphere 24 hafter transplantation of CellTrace-labeled BMMCs (red; A).Both IV and IA (n=4 for each group) treated animals showed sim-ilar numbers of BMMCs in the peri-infarct, as shown in the graph(B). Nuclei were visualized with Sytox green (green). Scale bar:20 μm.

Figure 5 Intravenous and intra-arterial administrations ofBMMCs showed a similar level of functional recovery in the cylin-der test. Analysis of the sensorimotor function in the cylindertest showed a significant recovery of the impaired forelimb inboth IA (n=5) and IV (n=8) groups compared with the salinegroup (n=5 IA, n=5 IV). This effect was maintained for11 weeks, corresponding to the period studied, and no signifi-cant difference was observed between the IA and IV groups.Data shown in the graph are means±S.E.M. (*) and (#) representcomparisons of the IV and IA groups respectively, with the salinegroups. ***=pb0.001, ##=pb0.01; Bonferroni post-test.

5Biodistribution and efficacy of bone marrow mononuclear cells after focal cerebral ischemia

2010; Kamiya et al., 2008; Schwarting et al., 2008). Becauseof the possible involvement of other organs, and mainly tocompare the distribution between the IA and IV routes, welabeled the BMMCs with 99mTc. Intense uptake in the headwas detected by the whole-body scintigraphies, showingthat transplanted cells reached some tissue either insidethe brain or in other surrounding structures. Surprisingly,scintigraphy analysis of the head and other organs after re-moval of the brain, revealed that the high uptake continuedin the head 2 h after the transplant. Analysis by gamma-wellcounting in the isolated organs confirmed that the high ac-tivity in the head observed in the whole-body images didnot occur in the brain of the animal that received IA injec-tion. Histological analysis after CellTrace labeling also indi-cated a small number of BMMCs in the peri-infarct area,after both IA and IV injections, whereas no cells werefound in non-ischemic animals. Moreover, analysis of the ra-dioactivity in the isolated organs indicated that IV and IA in-fusions led to similar distributions in the different organs inboth non-ischemic and ischemic animals. The only organthat showed a difference was the brain, where ischemic

animals had greater activity than non-ischemic animals inthe lesioned (left) hemisphere compared to the non-lesioned (right) hemisphere at 24 h, and this occurred afterboth IV and IA injections. These two pieces of data togethersuggest that a significant part of the transplanted cells mayhave homed to a tissue outside the brain but still withinthe head. A potential explanation is that since the injectionwas made in the common carotid artery, a significantamount of cells could have been distributed along the tissueirrigated by the external carotid artery, such as the naso-pharynx region. Importantly, 24 h after the transplant, thestrong signal was no longer present in the head of the IAgroup, indicating that the homing to this region was transito-ry. The amount of cells that migrated to the brain at 2 h and24 h was very small, which is in accordance with previousstudies (Detante et al., 2009; Gao et al., 2001) and suggeststhat cells may not need to remain in the brain to generatefunctional improvement. In a previous study, we demon-strated a therapeutic window for IV administration ofBMMCs up to 7 days after injury, with a better outcomewhen the cells were injected 1 day post-ischemia (deVasconcelos Dos Santos et al., 2010). In that report, thetreated animals reached a score of approximately 80% func-tional recovery. In an attempt to investigate whether the IAdelivery could overcome this effect, we evaluated this pro-cedure with IV administration, using the optimum time win-dow and dose identified in the previous studies. Our resultsshowed that there was no significant difference in the effi-cacy of the therapy between the animal groups that re-ceived IA and IV, and both showed functional improvementcompared with the control (saline-treated) group. Differentresults were reported by Kamiya et al. (2008). In their study,IA administration of BMMCs led to a decrease in total infarctvolume and improvement in motor function, as assessed witha rotarod test, when compared to the vehicle group, and this

6 A. Vasconcelos-dos-Santos et al.

effect was not seen in the IV group. The data in Kamiya andcolleagues' report were generated from the analysis of ani-mals in which transient ischemia was performed by middlecerebral artery occlusion (MCAO), differently from ourstudy, where the lesion was restricted to the cerebral cortexby thermocoagulation (TCL), which causes a permanent is-chemia. Additionally, the number of injected cells used inour model was 3 times higher than the number used byKamiya and colleagues, and the injection was performed24 h after the ischemia, instead of immediately after thetransient MCAO. Furthermore, Kamiya's group conductedthe histological analysis at an earlier time point, 1 h afterthe re-perfusion, and initially entrapped cells may no longerpersist at subsequent time points. These differences may beresponsible for the conflict in findings between our reports.

Conclusions

To our knowledge, this is the first study to compare IA and IVroutes of administration of BMMCs for the therapy of corticalischemia, produced in a model of thermocoagulation, interms of biodistribution and efficacy. Twenty-four hoursafter cell transplantation, homing in the brain seems to becomparable between both routes in ischemic animals as an-alyzed by 99mTc and CellTrace labeling, and greater thanthat of non-ischemic animals. Moreover, the functional stud-ies indicate a similar recovery of the sensorimotor function,as evaluated by the cylinder test, up to 11 weeks post-ischemia after IV or IA BMMC therapy.

Material and methods

Animals

Experiments were performed in adult male Wistar rats (3 to5 months old) weighing 250 to 450 g. Our experiments werecarried out in accordance with the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals(NIH Publication No. 80-23), and were approved by the Insti-tutional Committee for the Use of Experimental Animals(IBCCF 076).

Surgery

The ischemic lesion was induced by thermocoagulation ofthe blood in the pial blood vessels of sensorimotor cortices,as described previously (Szele et al., 1995). Briefly, the ani-mals were anesthetized with ketamine hydrochloride(50 mg/kg, i.p.) and xylazine hydrochloride (10 mg/kg,i.p.) and placed in a stereotactic frame. The skull was surgi-cally exposed and a craniotomy was performed, exposing theleft frontoparietal cortex (+2 to −6 mm A.P. from the Breg-ma). The blood in the pial vessels was thermocoagulatedtransdurally by bringing a hot probe close to the duramater. The color of the blood vessels is normally light red,and we considered the blood completely thermocoagulatedafter it turned to dark red. Care was taken to avoid touchingand tearing the dura mater. After the procedure, the skinwas sutured and the animals were kept warm under a heat

lamp and returned to the colony room after they recoveredfrom the anesthesia.

Obtaining BMMCs

BMMCs were isolated from the tibias and femurs of normalsyngeneic donor rats. Briefly, bone marrow was aspiratedfrom the bones and dissociated with serum-free DMEM-F12(GIBCO BRL, Grand Island, NY, USA) and collected in steriletubes. Bone-marrow cells were then mechanically dissociat-ed, centrifuged for 5 min (250×g), and resuspended in 4 mlof DMEM-F12. This volume was gently added over 4 m of His-topaque 1083 (Sigma-Aldrich, São Paulo, Brazil) and centri-fuged for 30 min (400×g). The cells were collected fromthe mononuclear cell layer and washed with phosphate-buffered saline (PBS), pH 7.4 in three consecutive series ofcentrifugations at 250×g (5 min). Following a final centrifu-gation, approximately 3×107 BMMCs were suspended in500 μl of saline and either injected immediately in the ani-mals submitted to the behavior test, or labeled for distribu-tion analysis.

BMMC labeling

BMMCs were labeled with 99mTc following protocols de-scribed previously (Barbosa da Fonseca et al., 2010;Battistella et al., 2011; Carvalho et al., 2008; Lassance etal., 2009; Quintanilha et al., 2008). Briefly, 500 μl of sterileSnCl2 solution was added to the cell suspension in phosphate-buffered saline (PBS), and the mixture was incubated atroom temperature for 10 min. Then, 5 mCi 99mTc wasadded and the incubation continued for another 10 min.After centrifugation (500×g for 5 min), the supernatantwas removed and the cells were washed three times withPBS. Viability of the labeled cells was assessed by the trypanblue exclusion test, and was estimated to be greater than93% in all cases. Labeling efficiency (%) was calculated bythe activity in the pellet divided by the sum of the radioac-tivity in the pellet plus supernatant, and was estimatedto be greater than 90% in all cases. Approximately 3×107

99mTc-BMMCs were injected in ischemic rats (1 day after is-chemia) and non-ischemic rats through the jugular vein (IV)and common carotid artery (IA), immediately after labeling.For histochemical analyses, the BMMCs were incubated withCellTraceTM Far Red DDAO-SE (Invitrogen) diluted in DMEM(1:500) for 45 min at 37 °C in a 5% CO2 incubator. Thesame amount of cells (3×107 99mTc-BMMCs) was injected inischemic animals by IV (n=4) and IA (n=4) routes after Cell-Trace labeling. Twenty-four hours after transplantation,the animals were euthanized and fixed with 4% paraformal-dehyde. Three sequential coronal slices were obtainedfrom three distinct antero-posterior regions (2.2, 1.0, and0.4 mm from the Bregma). CellTrace-positive cells werecounted in the photomicrographs obtained from the entireperiphery of the lesion, by confocal microscopy. The photo-micrographs were taken using a 20× objective, averagedacross the 6–7 photomicrographs in each of the slices.Counts were made in a double-blind manner, and the statis-tical analysis was done using GraphPad Prism 5.02(1992–2004 GraphPad Software, Inc.), using a paired t-test.

7Biodistribution and efficacy of bone marrow mononuclear cells after focal cerebral ischemia

Biodistribution analysis

Whole-body nuclear imaging was performed for qualita-tive biodistribution in a GE Millennium Gamma Camera (Gen-eral Electric Medical Systems, Milwaukee, Wisconsin, USA)equipped with a high-resolution collimator. A 15% energywindow centered on the 140 keV photo peak of 99mTc wasused. At 2 h and 24 h after transplantation, animals were di-vided into two major experimental groups, ischemic (n=6)and non-ischemic (n=4), receiving injection of 99mTc-BMMCs by IA or IV route, in a total of 8 groups (supplementalFigure 2). For quantitative biodistribution analysis, immedi-ately after whole-body nuclear imaging, the rats were eu-thanized and the left and right cerebral hemispheres,heart, lungs, liver, kidneys, spleen and stomach were re-moved and weighed. A scintigraphy of the isolated brain,heart, liver, spleen, kidneys, lungs, and stomach and of thehead without the brain was also performed in the ischemicanimals 2 h after cell injection. The total radioactivityinjected in each animal and the remaining radioactivity ineach organ were measured in a gamma counter (Cobra IIAuto-Gamma, Packard, USA). The percentage of the doseper organ [% dose/organ: each organ counts/total injectedactivity×100] and the injected dose (ID) per gram of tissue[%ID/g:% dose/organ/mass (g)] were determined for eachsample.

Behavioral testing

Functional recovery was evaluated using the cylinder test(Schallert, 2006; Schallert et al., 2001), which allows mea-surement of forelimb use asymmetry. The analyses wereperformed by blinded investigators to avoid bias. Animalsof all experimental groups were subjected to one trial onthe pre-ischemic day and then weekly until 77 post-ischemic days (PID). The trial consisted of placing the animalinside a glass cylinder (20 cm in diameter and 30 cm inheight). To prevent habituation to the cylinder, the numberof movements recorded in any one trial was limited to 20.The occurrences of sole use of the ipsilateral (to the lesion)or contralateral forelimb or the simultaneous use of bothforelimbs were counted. For each animal on each of the ex-amined PIDs, the percentage relative to the total number ofuses (ipsilateral+contralateral+simultaneous) was calculat-ed for the ipsilateral (unimpaired) uses and for the contra-lateral (impaired) uses. Then, one asymmetry score foreach animal was calculated on each PID by the following for-mula: asymmetry score=(% of ipsilateral uses)− (% of contra-lateral uses). Following this, the asymmetry score wasconverted to the symmetry score (100−asymmetry score).The animals submitted to functional evaluation were testedbefore the ischemia, 1 PID, when they received the trans-plant, and weekly until 11 weeks.

Statistical analysis

For statistical analysis of the behavioral tests, repeatedmeasures two-way ANOVA was used for comparison amonggroups. Because a significant interaction was observed inthe cylinder test, a Bonferroni post-test was performed for

each PID. The level of significance was always set atpb0.05. The biodistribution was analyzed using the pairedt-test.

Supplementary materials related to this article can befound online at doi:10.1016/j.scr.2012.02.002.

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