145ISSN 1746-075110.2217/RME.13.2 © 2013 Rosalia Mendez-Otero Regen. Med. (2013) 8(2), 145–155

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Biodistribution of bone marrow mononuclear cells after intra-arterial or intravenous transplantation in subacute stroke patients

Stroke is responsible for 9% of deaths world-wide, and is the second leading cause of death after ischemic heart disease [1]. Approximately a quarter of patients who suffer a stroke die within 1 month, and a half within 1 year [2]. Stroke is also a major cause of disability in the world, and with the progressive aging of the population, its incidence and consequently all social and eco-nomic burdens associated with it are expected to increase [2]. To date, the only pharmacologi-cal agent approved worldwide for use in patients with acute ischemic stroke is tissue plasminogen activator, but because of restrictions such as its narrow therapeutic window of 4.5 h, its use is limited to only a minority (2–4%) of patients [3]. The injury caused by stroke is mostly com-plete after 24–48 h, and neuroprotective therapies that must be administered within a time frame of 3–6 h have proven difficult to implement in clinical practice [4]. Alternatively, neurorestorative therapies aim to increase processes such as neu-rogenesis, angiogenesis and synaptogenesis [4,5], which could extend the time period for as much as 1 month or possibly longer, and could benefit large numbers of patients [4]. For this reason, cell therapy appears to be a promising therapeutic option for stroke. Cells of different origins, such as bone marrow mesenchymal stromal cells (MSCs) and bone marrow mononuclear cells (BMMNCs) have been applied in preclinical studies and have demonstrated beneficial effects in animal stroke models [4–6]. Small pilot clinical trials have also been performed to study the safety and possible

efficacy of cell transplantation in stroke patients. In this setting, noninvasive in vivo imaging tech-niques would be used to improve understanding of the different possible routes for cell delivery and their correlation with the functional evaluation of patients. In this study, we compared the biodis-tribution of autologous BMMNCs labeled with technetium-99m (99mTc) after intra-arterial (ia.) or intravenous (iv.) injection in 12 patients with sub-acute ischemic stroke. Our group has previously reported the clinical and imaging results of the first six patients from the ia. group [7–9].

Patients & methods�� Patients

This study, designed as a nonrandomized, open-label Phase I clinical trial, was approved by the Research Ethics Committee of our Institute and the National Committee of Ethics and Scien-tific Research, and registered in the NIH data-base (NCT00473057). All patients gave written informed consent. Inclusion and exclusion criteria are listed in Box 1.

The clinical and imaging results of the first six patients from the ia. group were described pre-viously [7–9]. In this report, one patient was added to the ia. group and five patients to the iv. group.

�� Bone-marrow aspiration, flow-cytometry ana lysis, cell separation & labeling with 99mTc On the day of the injection procedure, 80 ml of bone marrow was harvested from each patient

Aims: To assess the biodistribution of bone marrow mononuclear cells (BMMNC) delivered by different routes in patients with subacute middle cerebral artery ischemic stroke. Patients & methods: This was a nonrandomized, open-label Phase I clinical trial. After bone marrow harvesting, BMMNCs were labeled with technetium-99m and intra-arterially or intravenously delivered together with the unlabeled cells. Scintigraphies were carried out at 2 and 24 h after cell transplantation. Clinical follow-up was continued for 6 months. Results: Twelve patients were included, between 19 and 89 days after stroke, and received 1–5 × 108 BMMNCs. The intra-arterial group had greater radioactive counts in the liver and spleen and lower counts in the lungs at 2 and 24 h, while in the brain they were low and similar for both routes. Conclusion: BMMNC labeling with technetium-99m allowed imaging for up to 24 h after intra-arterial or intravenous injection in stroke patients.

KEYWORDS: 99mTc ��bone marrow mononuclear cells � cell transplatation � cerebral infarct � scinitigraphy

Paulo Henrique Rosado-de-Castro, Felipe da Rocha Schmidt, Valeria Battistella, Sergio Augusto Lopes de Souza, Bianca Gutfilen, Regina Coeli dos Santos Goldenberg, Tais Hanae Kasai-Brunswick, Leandro Vairo, Rafaella Monteiro Silva, Eduardo Wajnberg, Pedro Emmanuel Alvarenga Americano do Brasil, Emerson Leandro Gasparetto, Angelo Maiolino, Soniza Vieira Alves-Leon, Charles Andre, Rosalia Mendez-Otero*, Gabriel Rodriguez de Freitas & Lea Mirian Barbosa da Fonseca*Author for correspondence: Fax: +55 21 2280 8193 rmotero@biof.ufrj.br See pages 154–155 for a full list of affiliations

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and BMMNCs were separated by density gra-dient with Ficoll (Ficoll-Paque™ PLUS 1.077, 1:2, Amersham Biosciences, Brazil). After wash-ing, counting and viability testing, the cells were resuspended in 10 ml of saline solution with 5% autologous serum, and 2 × 107 cells were labeled with 99mTc based on previously published proto-cols [7,8,10]. In brief, 500 µl of sterile SnCl

2 solu-

tion was added to the cell suspension in 0.9% NaCl, and the mixture was incubated for 10 min at room temperature. Then, 45 mCi 99mTc was added and the incubation was continued for 10 min. After centrifugation (500 × g for 5 min), the supernatant was removed, the cells were washed again in saline solution, and the pellet was resus-pended in saline solution. The preparation and labeling of cells were all performed in a laminar flow. Bacteriology and culture were also carried out to exclude contamination of the material. A sample of the isolated BMMNCs was character-ized by flow-cytometry analysis of specific sur-face antigens as previously described [8]. Viability of labeled cells was assessed by the trypan blue exclusion test, and was estimated to be greater than 93% in all cases. Labeling efficiency was calculated by the activity in the pellet divided by the sum of the radioactivity in the pellet plus supernatant, and was estimated to be greater than 90% in all cases.

�� Cell transplantationLabeled cells were added back to the total mono-nuclear cell suspension (final volume of 10 ml). In the ia. group, femoral arterial punctures were carried out using a 6-Fr guiding catheter (Envoy-Cordis, FL, USA) or a Guider Softip™ (Boston Scientific, Target Therapeutics, CA, USA). iv. heparin was used for anticoagulation with an activated clotting time of two- to three-times the baseline. A digital cerebral angiography (Angio-star™, Siemens Medical Systems, Germany) was performed to allow visualization of the intracra-nial vasculature before infusion and monitoring of flow normality and vessel patency. A large-inner-diameter microcatheter (SL 1018 Boston Scientific, Target Therapeutics) was then navi-gated to the M1 portion of the middle cerebral artery. In the iv. group, cells were administered into the antecubital vein. In both groups the injection was performed at the rate of 1 ml/min.

�� ImagingScintigraphic scans were performed on a Mil-lennium™ GE camera (General Electric Medi-cal Systems, WI, USA). Images were acquired at 2 and 24 h after cell transplantation, with the patient in the supine position. Whole-body images were acquired for 20 min in anterior and posterior views with a dual-head whole-body

Box 1. Inclusion and exclusion criteria.

Inclusion criteria

� Age between 18 and 75 years

� Ischemic stroke in the middle cerebral artery territory within the last 90 days detected by computed tomography or MRI

� Recanalization of the middle cerebral artery that is involved as evaluated by transcranial Doppler studies or magnetic resonance angiography

� Score of 4–20 according to the NIH Stroke ScaleExclusion criteria

� Neurological decline (>4 points in the NIH Stroke Scale) before infusion, due to either edema or intracerebral hemorrhage

� Previous stroke with modified Rankin Scale >2

� Difficulty in obtaining vascular access for the percutaneous procedure

� Thrombophilias or primary hematological diseases

� Intracardiac thrombus

� Sepsis (according to the 1992 criteria of the Society of Critical Care Medicine and the American College of Chest Physicians)

� Autoimmune disorders

� Neurodegenerative disorders

� History of neoplasia or other comorbidity that could impact the patient’s short-term survival

� Bone disorders that could increase the risk of the bone marrow-harvesting procedure

� Liver failure

� Renal failure (creatinine >2 mg/ml)

� Life-support dependence

� Lacunar stroke

� Pregnancy

� Previous participation in other clinical trials

� Any condition that in the judgment of the investigator would place the patient at undue risk

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scanner with a high-resolution, low-energy col-limator. Planar images of the head were acquired for 10 min, using a 256 × 256 matrix in ante-rior, posterior, left lateral and right lateral views. Single-photon-emission computed tomography (SPECT) was performed at 2 h with two 180° opposed rotating detectors with low-energy high-resolution collimators. The software and hard-ware for image reconstruction was a Xeleris™ GE processing workstation for reconstruction of the SPECT image data. Each detector rotated 180° to allow a complete 360° revolution in a circular orbit, and projections were acquired over 24 min. The ordered subsets expectation maximization algorithm was used to reconstruct image volumes with axial smoothing and a Butterworth filter with an order of five and a cutoff of 0.45. Image volumes were composed of 64 × 64 × 64 voxels of 4.38 mm3 each. The detector distance varied from 14 cm to 20 cm during rotation, which resulted in a spatial resolution full width at half-max of approximately 12 mm. It was not possible to obtain SPECT images at 24 h for all patients due to the low count numbers at this time point.

Regions of interest were drawn for the brain, liver, lungs, spleen, kidneys and whole body, and the radioactive counts were automatically quan-tified for these regions in anterior and posterior whole-body planar images at 2 and 24 h after cell infusion. A geometric mean of anterior and pos-terior views was calculated to provide the organ-originated number of counts. In 2 h SPECT images, regions of interest corresponding to the left and right hemispheres were selected and the radioactive counts were automatically quanti-fied. The uptake in the hemispheres ipsilateral and contralateral to the lesion was defined as the percentage of hemisphere-originated counts com-pared with the total number of counts in both hemispheres.

�� Neurological evaluationAll patients were assessed by a board-certified neurologist at admission, on the day of cell transplantation and at 1, 7, 30, 60, 90, 120 and 180 days after cell injection, employing the NIH Stroke Scale (NIHSS), the Barthel Index and the modified Rankin Scale (mRS). Laboratory analyses (complete blood count and biochemi-cal tests for urea, creatinine and electrolytes) were also made at the same time points. A com-puted tomography (CT) or MRI was performed before the procedure and during the follow-up. An EEG was performed within 7 days of cell therapy and at any time if a patient’s neurological status worsened.

�� Statistical analysisThe analysis was mainly descriptive. The rank sum test was used to compare different variables between the ia. and iv. groups (age, NIHSS at admission, lesion volume, time from onset to injection, number of injected cells and uptake in different organs at 2 and 24 h). p-values lower than 0.05 were considered statistically signifi-cant. The R-project software [11] was used for data analysis.

Results�� Baseline characteristics

Twelve patients were included in the study, seven in the ia. group and five in the iv. group. The patient characteristics are listed in Table 1. Cell transplantation occurred between 19 and 89 days after the stroke, and the injected cell dose varied from 1 × 108 to 5 × 108 BMMNCs. The injected mononuclear fraction contained a median of 1.33% (range 0.56–2.48) hematopoietic stem cells (Ishage Protocol), 0.02% (range 0.01–0.04) MSCs and 0.01% (range 0.01–0.02) endothelial progenitor cells.

�� ImagingThe quantification of whole-body images indi-cated that the ia. route led to greater uptake in the liver and spleen and lower uptake in the lungs at 2 h compared with the iv. route (TaBle 2). In SPECT images, the ia. injection, compared with the iv. route, resulted in a greater uptake in the ipsilateral hemisphere at 2 h as compared with the uptake in both hemispheres (TaBle 2).

Quantifications performed 24 h after the transplantation showed that the iv. group had an increase in the percentage of uptake in the liver and spleen and a reduction in the lungs compared with whole-body scintigraphies at 2 h (TaBle 2). Uptake in the brain compared with the whole body remained low and similar between the groups at 2 and 24 h. Figures 1 & 2 show rep-resentative whole-body images from patients of the ia. and iv. groups, respectively.

�� Neurological evaluationBMMNCs were successfully injected without complications in all patients. No significant hemodynamic or clinical changes occurred dur-ing the injection. During the 6-month follow-up, serial clinical, laboratory and radiological evaluations showed no deaths or recurrence of stroke. During the follow-up, two patients of the ia. group and five patients of the iv. group had seizures that were controlled with

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antiepileptic medication. One patient (iv. 5) had neurological worsening after experienc-ing a partial seizure with secondary gener-alization at 111 days after cell therapy. The patient had an initial NIHSS of 11 that had decreased to 3 at 90 days after cell therapy, but after the seizure it returned to 11 (120 days) and later decreased to 8 (180 days) (Figure 3). NIHSS, Barthel Index and mRS scores can be found in supplemenTary TaBles 1, 2 & 3, respectively (see online www.futuremedicine.com/doi/suppl/10.2217/RME.13.2).

DiscussionDifferent experimental models of stroke have suggested that there is functional improvement after iv., ia. and intracerebral cell transplanta-tion of different cell types [12–19]. Among the many cell types tested, bone marrow-derived cells (MSCs and BMMNCs) have the advantage

of not raising the ethical issues that occur with fetal- and embryo-derived cells, and in addition are easily obtained from the patient for autolo-gous transplants, precluding the need for immu-nosuppression [4,6]. Another important advantage of bone-marrow cells is the existence of a large amount of safety information from clinical stud-ies with these same cells in malignant and non-malignant diseases [20,21]. Preclinical studies have demonstrated that they increase recovery in acute, subacute and chronic stroke, but it is not clear which cells are responsible for this effect, since improved function has been reported with dif-ferent cell populations [5,6].

Based on the data from preclinical studies, small Phase I and II clinical trials have been per-formed in stroke patients. Intracerebral injections of cells have been described for chronic stroke, using cell lines of immature neurons derived from a human teratocarcinoma (NT2N) [22,23], fetal

Table 1. Patient characteristics.

Patient Sex Infarct location

Risk factor Stroke subtype (according to TOAST)

Age NIHSS at admission

Lesion volume (cc)

Time from onset to injection (days)

No. of injected BMMNCs (×108)

ia. 1 M L PFO Cardioembolic 24 7 116 67 5.00

ia. 2 M R Hypertension, DM

Large artery 65 9 47 82 1.25

ia. 3 M L Hypertension Other† 47 4 17 62 3.90

ia. 4 M L AF, DM Cardioembolic 65 13 71 72 4.00

ia. 5 M R Hypertension Large artery 57 9 181 59 3.20

ia. 6 M R DM, hypertension

Large artery 47 13 213 73 1.00

ia. 7 M R DM, hypertension

Cryptogenic 60 12 2 19 1.30

iv. 1 M L AF, DM, hypertension

Cardioembolic 63 19 162 89 3.00

iv. 2 M L Hypertension Cardioembolic 63 16 106 89 5.00

iv. 3 M R Hypertension Cryptogenic 68 11 95 39 1.40

iv. 4 F R None Cryptogenic 39 15 144 20 1.50

iv. 5 F L DM, hypertension

Cardioembolic 57 11 39 25 5.00

ia. group

57 (47–62.5)

9 (8–12.5)

71 (32–148.5)

67 (60.5–72.5)

3.2 (1.3–4)

iv. group

63 (57–63)

15 (11–16)

106 (95–144)

39 (25–89)

3 (1.5–5)

Both groups

58.5 (47–63.5)

11.5 (9–13.5)

100.5 (45–148.5)

64.5 (35.5–75.2)

3.1 (1.4–4.2)

p-value 0.567 0.073 0.745 1 0.513

Results are expressed as median (interquartile range). The rank sum test was used to compare the ia. and iv. groups. †During aneurism clipping.AF: Atrial fibrillation; BMMNC: Bone marrow mononuclear cell; DM: Diabetes mellitus; F: Female; ia.: Intra-arterial; iv.: Intravenous; L: Left; M: Male; NIHSS: NIH Stroke Scale; PFO: Patent foramen ovale; R: Right; TOAST: Trial of Org 10172 in Acute Stroke Treatment.

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porcine cells [24] and BMMNCs [25]. No cell-related adverse effects were reported after stereo-tactical injection of the NT2N in 12 patients in Phase I or in 14 patients in Phase II, with strokes ranging from 6 months to 6 years previously [22,23], or after stereotactical injection of BMMNCs in five patients with ischemic or hemorrhagic strokes 3–8 years before therapy [25]. On the other hand, in the study that used fetal porcine cells intracer-ebrally injected in five patients with basal ganglia infarction 1.5–10 years before, two patients devel-oped seizures or aggravation of deficits and the study was halted prematurely [24]. Although intra-cerebral injection may lead to greater cell retention than intravascular delivery, it has the disadvantage of being an invasive procedure and leading to poor cell distribution through the lesion [16,26].

Furthermore, a growing body of preclinical evidence indicates that cells do not need to enter the brain to produce a therapeutic effect, and very few cells remain in the brain in preclinical studies that use iv. or ia. administration [4,6,27–29]. Instead of direct cell replacement, the functional effects of bone marrow-cell therapies seem to be mainly due to anti-inflammatory and immunomodulatory properties and the release of trophic factors such as brain-derived neurotrophic factor, NGF and VEGF [4,5,27]. In comparison with the delivery of solitary growth factors, cell therapy may provide the release of a variety of factors in a regulated manner [4,27].

Following a previously published preliminary report [30], Lee et al. [31] have recently described a clinical trial in which 16 patients received two iv. injections of MSCs at 4–5 and 7–9 weeks after stroke. In this study, patients were followed for up to 5 years and no significant side effects were observed. Furthermore, compared with the con-trol group, the mRS was decreased. Interestingly,

they found that clinical recovery in the MSC group was correlated with serum levels of stro-mal cell-derived factor-1a, which is upregulated in endothelial cells and astrocytes up to 4 months after stroke, and has been shown to direct the migration of bone marrow-derived cells [4,27,32,33]. The safety and feasibility of transplantation of

Table 2. Quantification of uptake in different regions at 2 and 24 h after cell transplantation.

Region 2 h 24 h

ia. iv. p-value ia. iv. p-value

Uptake in whole-body planar images (%)

Brain/whole body 0.8 (0.7–1) 0.8 (0.7–0.9) 1 0.6 (0.4–0.9) 0.9 (0.8–1) 0.254

Liver/whole body 40.6 (32.5–44.5) 13.5 (13.4–14) 0.006 46.9 (40.5–50.4) 18.5 (17.3–22.9) 0.006

Spleen/whole body 5.8 (5.6–6.2) 2 (1.7–2.2) 0.006 6.5 (5.4–7.8) 3.2 (3.2–3.4) 0.006

Lungs/whole body 7.1 (6.7–7.8) 20.9 (18.2–21.1) 0.006 4.3 (4.1–4.7) 7.8 (6.6–7.9) 0.006

Kidneys/whole body 4.2 (3.7–4.8) 3.2 (2.8–3.9) 0.329 7.6 (6.9–10.1) 7 (6.9–7) 0.255

Uptake in SPECT images (%)

Ipsilateral hemisphere/brain 68.1 (61.5–78.2) 53.4 (53.1–56.5) 0.023 NA† NA† NA†

Results are expressed as median (interquartile range). The rank sum test was used to compare the ia. and iv. groups. †It was not possible to obtain SPECT images at 24 h for all patients due to the low count numbers at this time point. ia.: Intra-arterial; iv. Intravenous; NA: Not available; SPECT: Single-photon-emission computed tomography.

Figure 1. Whole-body scintigraphies in anterior view of patient ia. 5. Whole-body scintigraph at (A) 2 h and (B) 24 h after cell transplantation. Increasing uptake is indicated by color shift from blue to red to yellow to white..

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MSCs for subacute ischemic stroke has been recently corroborated in another study that inves-tigated the potential of iv. injection of MSCs in 12 patients 36–133 days after the lesion [34].

One of the main disadvantages of using MSCs is the time needed to culture these cells (several weeks), which precludes the use of autologous cells in the acute phase of the stroke. In addition, the long culture time and the need for products of animal origin raises questions about safety in many countries. BMMNCs, on the other hand, do not require culture and may be obtained within hours of the stroke. The iv. injection of these cells has been recently performed in a trial with ten patients between 24 and 72 h after stroke, with no study-related adverse effects [35]. Likewise, Phase I clinical trials with ia. infusion of BMMNCs for acute ischemic stroke between 3 and 10 days after the infarct, which included a total of 35 patients, also indicated that the procedure is feasible and safe [36–40].

In one of the studies using BMMNC therapy for acute stroke, published by Moniche et al., two of ten patients had an isolated partial sei-zure during follow-up, and an antiepileptic drug

was started, with no seizure recurrence [40]. In our study, two patients from the ia. group (ia. 5 and ia. 6, at approximately 24 weeks after cell transplantation) and all patients of the iv. group (from 4 to 16 weeks after cell therapy) suffered seizures that were controlled pharmacologically. One of the patients of the ia. group (ia. 1) and one of the iv. group (iv. 1) had presented early seizures (<2 weeks after stroke) before enroll-ment. Although we cannot rule out the possibility that the incidence of seizures in this trial can be explained by chance and/or by the presence of risk factors, the occurrence of post-therapy seizures in seven of 12 patients (58%) warrants caution, and these patients were placed under extended follow-up. It has been estimated that the rate of late sei-zures (>2 weeks after stroke) varies from 3 to 67%, with great variation among series [41]. A number of risk factors have been associated with late sei-zures, such as cortical location, large infarcts and the occurrence of previous seizures [41–43]. Our patients had large, non-lacunar, middle cerebral artery infarcts, which may partially explain the high rate of seizures in the iv. group, which had greater stroke severity than the ia. group. Never-theless, it is also possible that the injected cells may activate brain repair mechanisms and modify the excitability in the peri lesional areas leading to sei-zures, and this point must be further investigated in future studies.

To assist in the understanding of the cell dis-tribution, we employed radioisotope labeling, a well-established technique that allows monitoring of cells in clinical practice [44]. Different preclini-cal and clinical studies in cell therapy have previ-ously used radioactive cell labeling as an indirect method to estimate homing intensity at different time points [10,45–50]. However, to our knowledge, no studies have reported the biodistribution of injected cells after iv. injection for stroke and com-pared it to ia. injection to date. In our current study, the labeling of cells indicated that the ia. route increased the number of radioactive counts in the ischemic hemisphere at 2 h in comparison with the total counts in both hemispheres after cell therapy, as quantified by SPECT. Nonethe-less, total uptake in the brain in comparison with the whole body was low, and similar between the ia. and iv. groups at 2 and 24 h. Interestingly, the greatest differences in uptake between the two groups were not found in the brain, but in other organs. The iv. group had lower radioactive counts in the liver and spleen and greater counts in the lungs at 2 and 24 h compared with the ia. group.

Few preclinical studies have compared ia. and iv. injections of BMMNCs, and their findings

Figure 2. Whole-body scintigraphies in anterior view of patient iv. 5. Whole-body scintigraph at (A) 2 h and (B) 24 h after cell transplantation. Increasing uptake is indicated by color shift from blue to red to yellow to white.

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differ according to the animal model and timing of injection. While Kamiya et al. [18] reported that ia. delivery led to greater brain homing and func-tional progress in comparison to iv. infusion in a model of transient ischemia, Vasconcelos-dos-Santos et al. [47] found that ia. and iv. injections promoted comparable functional improvement with low and similar brain homing in a model of permanent ischemia.

It has been previously demonstrated in animal models, that iv. cell injection leads to high uptake in the lungs [49,51–53]. A study in normal rats com-paring different cells found that the passage of BMMNCs was 30-fold higher than that of MSCs, and the differences in cell trapping were attributed to the smaller size and adhesion capabilities of the former [53]. While pulmonary activity may be seen as a negative factor because fewer cells reach arte-rial circulation, in an animal model of myocardial infarction, the trapping of MSCs in the lung led to secretion of TSG6, which decreased myocardial damage and increased myocardial function [4,54]. Likewise, the migration of cells to the spleen may also generate a therapeutic effect. After a stroke, immune cells in the spleen are stimulated to migrate to the brain, where they may contribute to tissue injury [55,56]. In animal models, it has been demonstrated that the iv. injection of hemat-opoietic stem cells after stroke may decrease the inflammatory reaction in the spleen and reduce the volume of the cerebral infarct [57].

Due to the short half-life of 99mTc of approxi-mately 6 h, we cannot rule out that the amount of

cells in the brain may increase at later time points. In a previous study using an animal model of stroke that employed green fluorescent protein bone mar-row hematopoietic stem cells, Schwarting et al. found that the number of cells in the brain after iv. injection increased at 72 h in comparison with 24 h, following early activity in organs such as the spleen [57]. Nevertheless, the number of cells in the brain was also small, even at 72 h, and estimated at approximately 0.02% of the injected cells [57]. Other radio pharmaceutical compounds such as Indium-111 oxine would allow monitoring for up to 96 h, but have disadvantages such as the interval of 18–24 h between infusion and imaging that is normally necessary, suboptimal photon energies, and a higher radiation burden for the cells and for the patient [44,58].

In addition to the drawback of not being able to follow the labeled cells for longer periods, this study had a number of limitations, such as the absence of a control group. The sample number was small, as approved by the National Commit-tee of Ethics and Scientific Research for an intro-ductory trial. Moreover, the iv. group had a higher stroke severity than the ia. group. This occurred because the inclusion of the iv. group was author-ized after the inclusion of the ia. group and the study was nonrandomized. Consequently, the difference in stroke severity limits the compari-sons between both groups. It is also important to indicate that due to the sample size and the absence of a control group, this study did not aim to evaluate the efficacy of cell therapy, and any

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Figure 3. Neurological evolution according to the NIH Stroke Scale. Neurological evolution after cell transplantation for the (A) ia. group and the (B) iv. group. ia.: Intra-arterial; iv.: Intravenous; NIHSS: NIH Stroke Scale.

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improvement in neurological scores observed in this small group of patients could be attributed to spontaneous recovery that may occur after stroke. Therefore, randomized controlled stud-ies are needed in order to avoid such limitations and to allow the investigation of the impact of factors such as the number of injected cells and the route and timing of delivery. Furthermore, larger randomized controlled studies will also improve the understanding of the importance of different types of deficits and stroke severities in the outcome of the treatment.

ConclusionLabeling BMMNCs with 99mTc allowed imaging for up to 24 h after ia. or iv. injection. Although there were greater radioactive counts in the ischemic hemisphere compared with the con-tralateral hemisphere at 2 h in the ia. group, total uptake in the brain compared with the whole body was low and similar between the two routes at 2 and 24 h. The greatest differences were observed in other organs, with the iv. route demonstrating lower uptake in the liver and spleen and higher uptake in the lungs at both time points.

Future perspectivePreclinical studies indicate that the transplanta-tion of stem/progenitor cells lead to structural and functional improvements in animal models of stroke. However, until now only small clini-cal trials using cell therapies for stroke have been published, indicating that they seem to be feasi-ble and safe. Therefore, there is a need for larger,

randomized controlled clinical trials to investigate whether these therapies are effective. Furthermore, other questions such as the cell type, route and timing of administration need to be investigated.

Financial & competing interests disclosureThis study was supported by a grant from the Ministry of Health and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) to R Men-dez-Otero (PP SUS-2009 grant 110.776/2010). PH Rosado-de-Castro and E Wajnberg received a PhD Scholarship from Coordenação de Aperfeiçoamento de Pes-soal de Nível Superior (CAPES). TH Kasai-Brunswick and L Vairo received PhD Scholarships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). RC Goldenberg, EL Gasparetto, A Maiolino, B Gutfilen, LM Barbosa da Fonseca and R Mendez-Otero received Research Productivity Scholarships from CNPq. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Writing assistance was used in the production of this manuscript. The authors thank JW Reid for revising and editing the language in the text. This was funded by the grant to R Mendez-Otero.

Ethical conduct of research The authors state that they have obtained appropriate insti-tutional review board approval or have followed the princi-ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investi gations involving human subjects, informed consent has been obtained from the participants involved.

Executive summary

Current burden of stroke

� Stroke is responsible for 9% of deaths worldwide, and is the second leading cause of death after ischemic heart disease.

� Stroke is a major cause of disability in the world.

� The only pharmacological agent approved worldwide for acute ischemic stroke is the tissue plasminogen activator, but its narrow therapeutic window of 4.5 h limits its use to only a minority (2–4%) of patients.

Findings from animal models & current clinical trials

� Animal models of stroke indicate structural and functional improvement after intravenous, intra-arterial and intracerebral cell transplantation of different cell types.

� Bone marrow-derived mesenchymal stem cells and bone marrow mononuclear cells do not raise the ethical issues that occur with fetal- and embryo-derived cells, and may be obtained for autologous transplants, precluding the need for immunosuppression.

� Experimental studies have demonstrated increased recovery in acute, subacute and chronic stroke, but it is not clear which cells are responsible for this effect.

� Noninvasive in vivo imaging techniques allow the comparison of the biodistribution of these cells after injection by different routes.

� Small pilot clinical trials have indicated the safety and possible efficacy of cell transplantation in stroke patients.

Results

� In this study, total cell homing in the brain compared with the whole body was low and similar between the intra-arterial and intravenous routes at 2 and 24 h.

� The intravenous injection led to lower uptake in the liver and spleen and higher uptake in the lungs at both time points in comparison with the intra-arterial infusion.

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ReferencesPapers of special note have been highlighted as:�� of considerable interest

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��� The first clinical trial of cell therapy for patients with stroke.

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��� The first clinical trial using autologous bone marrow mesenchymal cells in ischemic stroke.

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ReseaRch aRticle Rosado-de-Castro, Schmidt, Battistella et al.ReseaRch aRticle

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��� The first clinical trial using autologous bone marrow mononuclear cells in ischemic stroke.

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Affiliations � Paulo Henrique Rosado-de-Castro

Department of Radiology, School of Medicine, Universidade Federal do Rio de Janeiro, Rua Professor Rodolpho Paulo Rocco 255, Cidade Universitária, Ilha do Fundao, 21941-913, Rio de Janeiro, Brazil.

� Felipe da Rocha Schmidt Neurology Service, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

� Valeria Battistella Neurology Service, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

� Sergio Augusto Lopes de Souza Department of Radiology, School of Medicine, Universidade Federal do Rio de Janeiro, Rua Professor Rodolpho Paulo Rocco 255, Cidade Universitária, Ilha do Fundao, 21941-913, Rio de Janeiro, Brazil.

� Bianca Gutfilen Department of Radiology, School of Medicine, Universidade Federal do Rio de Janeiro, Rua Professor Rodolpho Paulo Rocco 255, Cidade Universitária, Ilha do Fundao, 21941-913, Rio de Janeiro, Brazil.

� Regina Coeli dos Santos Goldenberg Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho 373, CCS, Bloco G, Cidade Universitária, Ilha do Fundao, 21941-590, Rio de Janeiro, Brazil

� Tais Hanae Kasai-Brunswick Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho 373, CCS, Bloco G, Cidade Universitária, Ilha do Fundao, 21941-590, Rio de Janeiro, Brazil

� Leandro Vairo Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho 373, CCS, Bloco G, Cidade Universitária, Ilha do Fundao, 21941-590, Rio de Janeiro, Brazil

� Rafaella Monteiro Silva Neurology Service, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

� Eduardo Wajnberg Department of Radiology, School of Medicine, Universidade Federal do Rio de Janeiro, Rua Professor Rodolpho Paulo Rocco 255, Cidade Universitária, Ilha do Fundao, 21941-913, Rio de Janeiro, Brazil

� Pedro Emmanuel Alvarenga Americano do Brasil D’Or Institute for Research and Education, Rio de Janeiro, Brazil

� Emerson Leandro Gasparetto Department of Radiology, School of Medicine, Universidade Federal do Rio de Janeiro, Rua

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Professor Rodolpho Paulo Rocco 255, Cidade Universitária, Ilha do Fundao, 21941-913, Rio de Janeiro, Brazil

� Angelo Maiolino Hematology Service, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

� Soniza Vieira Alves-Leon Neurology Service, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

� Charles Andre Neurology Service, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

� Rosalia Mendez-Otero Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho 373, CCS, Bloco G, Cidade Universitária, Ilha do Fundao, 21941-590, Rio de Janeiro, Brazil

� Gabriel Rodriguez de Freitas Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,

Avenida Carlos Chagas Filho 373, CCS, Bloco G, Cidade Universitária, Ilha do Fundao, 21941-590, Rio de Janeiro, Brazil and D’Or Institute for Research and Education, Rio de Janeiro, Brazil

� Lea Mirian Barbosa da Fonseca Department of Radiology, School of Medicine, Universidade Federal do Rio de Janeiro, Rua Professor Rodolpho Paulo Rocco 255, Cidade Universitária, Ilha do Fundao, 21941-913, Rio de Janeiro, Brazil