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Therapeutics with SPION-labeled stem cells for the main diseases related to brain aging: a systematic review

The increase in clinical trials assessing the efficacy of cell therapy for structural and functional regeneration of the nervous system in diseases related to the aging brain is well known. However, the results are inconclusive as to the best cell type to be used or the best methodology for the homing of these stem cells. This systematic review analyzed published data on SPION (superparamagnetic iron oxide nanoparticle)-labeled stem cells as a therapy for brain diseases, such as ischemic stroke, Parkinson’s disease, amyotrophic lateral sclerosis, and dementia. This review highlights the therapeutic role of stem cells in reversing the aging process and the pathophysiology of brain aging, as well as emphasizing nanotechnology as an important tool to monitor stem cell migration in affected regions of the brain.


Brain aging
Brain aging is a very complex biological process associated with a decline in sensory, motor, and cognitive function. Aging is not a disease, it is a regular physiological process that can be developed without associated diseases. When this occurs, the process is referred to as "successful aging". 1 Aging brings the possibility of developing dementia as a result of the normal aging process, as has been postulated. 1 It has been suggested that in an individual aged 120 years old without associated disease, the neocortical synapses would be reduced to the same levels found in Alzheimer's disease (AD) with a 40% loss of intracerebral connectivity. This may result in senile dementia without amyloid plaques and neurofibrillary tangles, which characterizes AD. 2 Although these data are controversial and clearly refer to very old age, which in fact lies at the upper limit of human longevity, they reveal the close relationship between aging and disease.
The successes of public and individual health achievements have led to a large increase in human life expectancy. In the early 1900s life expectancy was about 45 to 50 years; today, people aged 100 years or older are no longer considered unusual, and in some nations represent 2% of the population. 3 This recent change in life expectancy has increased the number of elderly individuals.
The greater the number of older subjects living, the more individuals will experience changes associated with aging. In this sense, the incidence of diseases related to increasing age has also risen, especially stroke and neurodegenerative diseases, such as dementia, Parkinson's disease, and amyotrophic lateral sclerosis (ALS), which together affect approximately 3% of the population older than 65 years. 4 Thus, it is important to understand the mechanisms underlying longevity in human beings as well as the complex factors that make humans more vulnerable to neurodegenerative diseases. It is also important to study the therapeutics for restoring body and brain health during aging. 5,6 Stroke Ischemic stroke is described as a sudden death of brain cells in a concentrated area, caused by an interruption of blood flow. This disease represents the third leading cause of death and the first cause of disability in developed countries. In addition, it is a neurological disease with a high mortality; 6.4 per 100,000. 7 There are different subtypes of stroke: ischemic stroke, caused by occlusion (temporary or permanent) of a blood vessel supplying the brain, and hemorrhagic stroke, caused by rupture of a blood vessel in the brain. Ischemic stroke is the most common type of stroke, accounting for 85%-90% of cases compared with hemorrhagic, 10%-15%.
Thrombosis, embolism, or hypoperfusion may result in a restriction of blood flow to the brain, and may cause ischemic stroke, resulting in insufficient oxygen and glucose supply to support cellular homeostasis. This triggers several processes resulting in cell death by cytotoxicity or ionic imbalance. These processes lead to a significant increase in glutamatergic transmission, and as a consequence oxidative stress, inflammation, and apoptosis [8][9][10] (Figure 1).
Currently, the main treatment for neural damage limitation in patients with ischemic stroke is the early administration of thrombolytic agents, but their use is often limited by a narrow therapeutic time window. 8,11 Preclinical studies have been showing functional recovery after cell therapy. [12][13][14][15] Parkinson's disease Parkinson's disease is the second most common neurodegenerative disorder. The prevalence of this disease in industrialized countries affects roughly 0.3% of men, rises to 1% in the population older than 60 years and up to 4% in the population over 80 years. The mean age at diagnosis is around 60 years old, but 10% of cases classified as "early onset" show clinical symptoms in individuals between 20 and 50 years old.
Parkinson's disease affects multiple neurotransmitter systems and its cause is still unknown. The most accepted mechanism considered responsible for the classic motor features of the disease (bradykinesia, rigidity, tremor, and postural instability) is loss of dopaminergic neurons. These neurons are located in the substantia nigra and their degeneration leads to subsequent loss of endings in the basal ganglia (caudate, putamen [striatum], globus pallidus and subthalamic nucleus; Figure 1).
The treatments available nowadays provide only transitory symptomatic relief. In addition, there are serious complications that arise from chronic treatment with existing drugs. For example, the incidence of dyskinesia induced by levodopa is around 30%-50% after 5 years, and 60% after 10 years of treatment. 16,17 Pre-clinical studies which had used stem cell-based therapy (by 6-OHDA [6-hydroxydopamine], MPTP [(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)] or rotenone) showed functional improvement without dyskinesia induction. [18][19][20][21][22][23][24][25] Multiple sclerosis and ALS Multiple sclerosis (MS), also called "disseminated sclerosis" is characterized by degeneration of the myelin sheath surrounding the brain and spinal cord axons, 26 as shown in Figure 1. The main symptoms of axonal injury are characterized by motor and cognitive impairment. 27 ALS, also known as Lou Gehrig's disease or Charcot, is a progressive and fatal neurodegenerative disease that affects 2.16 of 100,000 individuals. ALS is not a demyelinating disease, but rather characterized by degeneration of motor neurons (mainly pyramidal fasciculus in the lateral funiculus of the white matter of the spinal cord) that coordinate voluntary muscle movements observed in the clinical setting, which generate denervation by atrophy, such as loss of muscle mass with progressive difficulty in performing movements (akinesia), and muscle strength loss (atony). 27 The actual arsenal of therapies available promotes delay/ blocks the process without restoration of functional loss in MS. However, in ALS the situation is more dire, because on average patients die 4 years after being diagnosed. 28 Functional improvement was seen in preclinical [29][30][31][32][33] and clinical studies 34 after cell therapy.

Dementias
Dementia is a progressive deterioration of cognitive functions, such as memory, language, and learning. 35 Some neurodegenerative diseases such as Huntington's and Parkinson's disease can develop into dementia. 36 However, AD is still the leading cause of dementia in elderly people, accounting for 60%-80% of cases. 37 AD is a progressive neurodegenerative disease that can affect 60% of seniors over 80 years old. 38 The cause of this disease remains unknown, but it is notorious that genetic 3751 SPION-labeled stem cells for the diseases related to brain aging changes are found in less than 1% of cases. 39 AD is associated with age and is characterized by deterioration of cognitive abilities, initially established by changes in recent memory. Disturbances in later stages of AD result in a generalized cognitive impairment, and patients become completely dependent on care givers' support. These stages often culminate in death. 39 The brain of a patient who developed AD is shown in an illustrative manner in Figure 1G. This disease is characterized by the accumulation of senile plaques in the extracellular space of the cerebral cortex and hippocampus. Essentially, the plaque contains a small peptide called amyloid-beta (Aβ). 40 AD is also characterized by intracellular neurofibrillary tangles shaped by hyperphosphorylated Tau protein, 41 and reduced levels of nicotinic receptors (nAChRs) in regions that present protein aggregation. 42 Stem cell implications in diseases related to brain aging Stem cells are characterized by their ability to divide and give rise to identical daughter stem cells, and to differentiate into other cell types. 43 Due to these features, stem cells are important during embryonic development for tissue formation and during adulthood to ensure tissue homeostasis, repair, and regeneration. [44][45][46] The physiological loss of tissue homeostasis during life is typically connected to a progressive and extensive decline in the physical and cognitive performance of the whole organism, commonly referred to as aging. 47,48 In addition to loss of stem cell turnover, aging is also caused by a decrease in the overall function of differentiated cells, which occurs due to the cell number and environmental factors including DNA damage, reduction in telomere length, oxidative stress,

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Alvarim et al induction of stress response pathways, and production and accumulation of misfolded proteins. [49][50][51][52][53][54][55] These functional changes may result in a number of disorders including neurological diseases and many other deleterious effects of age on the genome, epigenome, and proteome. Some of the functional changes arise in the cells autonomously and others are imposed by an age-related change in the local milieu or systemic environment. Notably, some changes, particularly epigenomic and proteomic, are potentially reversible, and both environmental and genetic interventions can result in the rejuvenation of aged stem cells. 53,55 Such findings have profound implications for stem cell-based therapy of age-related diseases.

Nanobiotechnology: possibilities
Cell therapy is presented as a new therapeutic strategy, mainly when standard therapy is ineffective, but interpretation of results is often biased. A major challenge is the lack of consistent data on stem cells' survival, distribution, and repair process leading to functional recovery in in vivo experiments. 56 To address these issues, a method of noninvasive cellular analysis is more efficient than conventional histopathological techniques, and provides unique information about cell behavior over time. 57 In this sense, several imaging modalities are available, including computerized tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), computed tomography photon emission tomography (SPECT), optical imaging, and ultrasound. The advantages of MRI compared to other technologies include its high spatial resolution and no exposure of animals or patients to ionizing radiation, which is needed in CT, PET, and SPECT. 58,59 The MRI "homing" analysis method of transplanted stem cells occurs by the use of contrast agent. Nowadays, several available magnetic nanoparticles exist, 56 and the most commonly used one is iron oxide. 60 The processes by which nanoparticles are internalized by cells are not fully elucidated; however, mechanisms of endocytosis well described in the literature are pointed out as the main routes of internalization, including: i) internalization mediated by membrane receptors, ii) internalization independent of clathrin and caveolin, iii) clathrin-mediated internalization, iv) internalization mediated by caveolin, and v) transfection agent mediated internalization by poly-L-lysine (PLL), as shown in Figure 1.
It is necessary to understand how these endocytic processes can affect cellular homeostasis, including signal transduction, organization, and cell viability. 60 Though, due to this cellular capacity, nanostructures can be inserted into a complex organism allowing cellular viewing, location, and tracking in vitro and/or in vivo.
Therefore, nanobiotechnology can offer many benefits to stem cell research, including cell tracking, which is currently the limiting factor for further advances in this field of science.
The aim of this review was to analyze publications on SPION (superparamagnetic iron oxide nanoparticle)labeled stem cells and their application in major diseases related to the aging brain, such as ischemic stroke, Parkinson's disease, sclerosis and dementias such as AD.

Data compilation and review
In this review, five of the authors independently and randomly selected (in pairs) applied each disease using search strategies in the databases cited above.

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SPION-labeled stem cells for the diseases related to brain aging MFA, and LPN for MS and ALS; LTA, EADB, and LPN for AD and dementias. The analysis process and table plots were carried out by full consensus of peers, respecting the distribution above. In cases of disagreement, a third, independent author decided to add or subtract data.
Subsequently, JBM and LFG reviewed nanoparticles' characteristics, HRS and LTA stem cells' characteristics, LPN and LTA animal model characteristics and in vivo assay, finally LPN, LFG, and MPN neuroimaging methods used to assess therapeutic efficacy. In cases of disagreement, a third, independent author decided to add or subtract data.

Selection criteria
This review included only original articles written in English that had used SPION-labeled stem cells (SC/SPION) in preclinical models of stroke, Parkinson's disease, MS, ALS or AD and other dementias, published between 2000 and 2013.
Publications indexed in more than one database (duplicates), incomplete, conference presentations, book chapters, and non-English, as well as those that had not used SPION or SC were excluded from this review ( Figure 2).

Results
After applying search strategies, 86 original articles were identified after expulsing the duplicated articles with selection of database. Of these, 44 publications were excluded for not including SPION and/or SC, nine were reviews, and one study 61 which was written in Chinese. A total of 32 original articles satisfied the inclusion criteria: 20 on ischemic stroke, seven on Parkinson's disease and five on sclerosis ( Figure 2).

Total number of articles identified in searches N=86
-    14,74 used Sinerem ® one study 72 used FluidMag-D ® , FluidMag-lipid ® , DEAE-FluidMag ® , FluidMag-P ® and FluidMag-Q ® (all from Chemicell GmbH, Berlin, Germany), and another study 64 used iron fluorescent nanoparticles (excitation 480 nm and emission 250 nm). In addition, 13 studies evaluated commercial nanoparticles, four studies evaluated nanoparticles synthesized by the group itself ("in situ"): i) Lee et al 13 used nanoparticles synthesized from polymerization and coprecipitation processes. These processes were modified to obtain nanoparticles of different diameters 100-750 nm; ii) Reddy et al 62 used magnetic nanoparticles synthesized by sonochemistry method, followed by chitosana coating; iii) in a study by Wang et al 63 the synthesis occurred in two steps: first, synthesis generated polystyrene nanocluster magnetite (PMNC) and second, it promoted PMNC coating with a layer of silica and rhodamine; iv) Lu et al 75 69 another study 24 used Resovist ® . Besides these, two studies 19,21 synthesized the nanoparticles ("in lab"). Bjorklund et al 19 synthesized magnetite core coated with dipalmitoyl phosphatidic acid sodium (DPPA-Na) and ethylene oxide propylene oxide block copolymer (F108 ® ; Bracco Research, Geneva, Switzerland). Jackson et al 21    The majority of superparamagnetic iron oxide nanoparticles are coated by dextran (dextran T10 and carboxydextran) as identified in 21 articles. [12][13][14]20,22,24,31,32,65,[66][67][68][69][70][71]74 In addition to dextran, seven other types of SPION coatings were identified, such as starch, phosphatidylcholine, DEAE-diphosphate starch and poly-diallyl-dimethylamine in the Riegler et al 72 study; carboxylated iron oxide core was seen in the Detante et al 64 study, silica in the study by Wang et al 63 monomer citrate in the study by Stroh et al 23 and DPPA in the study by Bjorklund et al. 19 There was one study 62 that used both dextran and chitosan.

synthesized iron oxide
The physico-chemical features of the SPION used the most are: i) Feridex ® also known as Endorem™, iron oxide nanoparticles with magnetite crystal phase, and mean core measuring 5-6 nm in diameter, and hydrodynamic diameters of 50-180 nm, coated with dextran 10 and provided in an iron concentration of 11.2 mg/mL; ii) Resovist ® , iron oxide nanoparticles with magnetite crystal phase and maghemite core measuring on average 4 nm in diameter, and hydrodynamic diameters of 55-65 nm, coated with carboxydextran and provided at an iron concentration of 27.9 mg/mL; iii) Sinerem ® , iron oxide nanoparticles with magnetite crystal phase, core measuring 5-6 nm in diameter, and hydrodynamic diameters of 20-40 nm, coated with dextran 10 and provided at an iron concentration of 20.0 mg/mL.

In vitro experiments for stem cell labeling
Regarding the analysis of the SPION cell labeling process by the 32 selected articles, the majority (19) Table 2).
The use of a transfection agent and a culture medium to optimize the stem cell labeling process with SPION is usual. In 15 publications the transfection agent was used, of these studies, 12 used PLL and Dulbecco's Modified Eagle's Medium (DMEM) as their culture  15,23,30,31,67,69,70,75 of which 16 studied some method of analysis, using the MTT assay and/or trypan blue, and also testing cells' differentiation into mesodermal lineages as osteogenic, adipogenic, and chondrogenic in five of the 32 studies. 12,14,62,64,73 After analysis of these parameters for each disease related to brain aging, a small variation between the sources was observed, cell types, and methods for SPION labeling.

Parkinson's disease
Out of a total of seven selected studies on SPION labeled stem cells in Parkinson's disease experimental model (Table 4), three studies 21,22,24 used MSC. Of these three studies, two used bone marrow as a source of MSC. 22,24 Two of the remaining four articles used neural stem cells 20,25 and two used embryonic stem cells. 19,23 One study 22   Only one study 23 reported the method used to determine cell viability (Annexin V/propidium iodide). No studies mentioned the relative value of SPION cell labeling efficiency.

Multiple sclerosis and ALS
Only five studies [30][31][32][33][34] (Table 2) were selected that used cells from different sources, such as: stem cells derived from human skeletal muscle -SkmSCs, 30 bone marrow derived human MSC, 34 amniotic fluid derived from human stem cells, 32 human embrionary stem cells, 33 and neural rat stem cells. 31 To analyze the culture mediums, two studies 32,34 used basic DMEM, and one study 30 supplemented the medium with epidermal growth factor (EGF; 20 ng/mL), basic fibroblast growth factor (bFGF; 10 ng/mL), neural stem cell growth N2 and Noggin (100 ng/mL), and Kim et al 33 used only DMEM supplemented with stem cell N2.
The cell concentration used in these studies ranged from 10 5 to 10 6 cells. All selected studies used commercially available particles: Endorem™, Feridex ® , Resovist ® , Molday ION, and Sinerem ® , as described in the previous discussion (see Physico-chemical characteristics of SPION section). Only two studies 31,32 identified SPION concentrations ranging from 35 μg/mL to 1,000 μg/mL. Related to the incubation period, one study 34 used ranged intervals, 24-48 hours for labeling, and two other studies 31,32 set a time of 72 hours.
For cell viability, one study 30 used caspase 3, TUNEL assay method of analysis, and another study 32 used only MTT assay. Regarding the efficiency of cell labeling, the studies 30-32 only used the term "good labeling", and two studies 31,32 reported 100% efficiency.
In the clinical study selected, 34 MSC labeled with Feridex ® (Advanced Magnetic) injected intrathecally or intravenously led to immediate immunomodulatory effect, and promoted axonal demyelination.

In vivo experiments -human trial or animal models with SPION/SC
Of the 32 studies selected in this review, only two were clinical trials; 34,65 the remaining 30 studies used experimental or pre-clinical models. Among those, one study 75 used a dog, two studies 62,72 used rabbits, 27 studies used rodents, especially Sprague-Dawley rats, and only five studies 30-32,63,73 used mice. This profile suggests that the use of SPION labeled stem cells for cell therapy with regards to aging brain diseases is still in the initial phase, see Figure 3. Positive aspects and limitations for this therapy will be analyzed for each in vivo experiment.
Only five studies 12,70,71,75,76 conducted behavioral tests, adapted from clinical neurology scales. The remaining studies evaluated the model effectiveness through various parameters of MRI and magnetic fields, which will be described in the next section.
The procedure of grafting stem cells with SPION (SC/ SPION) was performed using both intracerebral application in specific regions, such as striatum 12,63,64,67,68,70,73,74 hippocampus 67 and corpus callosum, 74 or nonspecific regions, such as intraventricular/cortical, 13,15,62,66,75 as applied in the peripheral vascular bed 66,69-72 (femoral artery) or cerebral vascular bed 15,69,71 (internal carotid artery). The average time between the ischemic event and therapy with SC/ SPION was roughly 3.43 days or 82 hours. However, some studies 15,71,72,75 underwent implantation immediately after stroke, and another study 74 performed the therapy 14 days after the ischemic event took place. The most significant data found were the functional recovery after 14 days of SC/SPION implantation described by Gutiérrez-Fernandéz et al's study. 71 The clinical trial 65 included was composed of 205 patients aged around 71 years with history of ischemic stroke in the past 3 to 31 days with National Institutes of Health Stroke Scores (NIHSS, 10.4±6.5) and percentage thrombolytic medication use at admission (6.4%±1.2%). This study used adverse effects as a primary outcome, including death, and as a secondary endpoint they assessed motor and cognitive aspects by NIHSS scores, modified Rankin score, the index of Nottingham Extended Activities of Daily Living, the Barthel index, the Mini-Mental State Examination and the Zung depression scale, before and after 10, and 90 days of the 10 6 granulocyte-colony stimulating factor (G-CSF) cells' administration. No significant change was found in the functional scale. A slight improvement was seen in the NIHSS scores and the modified Rankin score. When performing a meta-analysis together with three other studies on this topic an odds ratio of 1.11 was observed, suggesting a low potential benefit for results presented.

Parkinson's disease
All studies used experimental models based on rodents (Table 4). Of the seven studies [19][20][21][22][23][24][25] which composed this sample, only one study 23 used Wistar rats, the other studies used Sprague-Dawley rats. The average weight of the animal was 250 g, and the number of animals per group ranged from 12-63 divided into 2-6 groups.
The included studies had control groups, and they infused and compared the medium (DMEM) with the group that received stem cells. The disease induction model used in almost all these studies was the 6-OHDA. Animals were submitted to the drug dependent rotation test (dopaminergic agonist: apomorphine or amphetamine), which was performed more than 20 full turns per minute, for at least 10 minutes. This behavioral test was performed at different periods of time depending on the article; some studies 21,22,24 applied the test only once after the neurotoxin 6-OHDA administration, but one study 19 submitted animals to the test several times for 9 weeks. The average observation time was 4 weeks or 28 days before stem cell infusion. The induction model was neurotoxin locally administrated in the medial forebrain bundle (MFB); two studies 21,22 used intrastriatal injection of neurotoxin.
The mean time after induction of the disease model for stem cell infusion was 4 weeks or 28 days. All studies infused stem cells in different areas within the striatum region as shown in Table 4. The median number of infused cells was around 10 5 cells/3 μl, and the animals' observation time after treatment was on average approximately 6.3 weeks or 42 days. Most of these studies used amphetamine ("primer") to verify dopaminergic imbalance inherent to the process of coordination and central motor adjustment. 19

Multiple sclerosis and ALS
There were five studies on stem cells labeled with SPION used in MS or ALS. There was one clinical article, 34 and the other four articles [30][31][32][33] (Table 5) used experimental models of genetically modified mice to generate autoimmune encephalomyelitis, called Wobbler (Wr). Animals heterozygous for the gene Wr used in studies were 4 weeks 31,32 to 10 weeks 33 old; they underwent implantation of SC/SPION intraventricularly at concentrations ranging from 10 5 in the Bigini et al 31 study to 10 6 in two other studies. 30,33 The Bigini et al 31 study was the one that only used immunosuppressive therapy (cyclosporine 15 mg/kg daily) during the 28 days of the experiment. After implantation of SC/SPION, these studies monitored animals by neuroimaging using the MRI technique from SC/SPION implantation and up to 28 days, 31 and the average time of monitoring was 9 days. Other parameters of neuroimaging studies will be addressed in the next section, Table 5.
The clinical trial 34 phase one and two, recruited 15 patients with MS and 19 patients with ALS. The mean age of patients with MS was 35 years; they also had a history of disease for about 10 years. ALS patients' mean age was 53 years and their history of disease was about 34 months. Functional scales, EDSS for MS and ALSFRS for ALS were used for therapeutic monitoring, besides the MRI, which will be discussed in the next section. MS patients received 2.5×10 6 MSC intrathecally. ALS patients also received 6.0×10 6 MSC intrathecally. Patients were monitored for MSC immunoregulatory behavior for 4, 24, and 48 hours after implantation. Neuroimaging monitoring occurred at the same periods mentioned above, as well as 1, 3, and 6 months after implantation. A significant improvement in clinical conditions for both diseases, more evident in MS, was observed using the functional scales previously International Journal of Nanomedicine 2014:9 submit your manuscript | www.dovepress.com

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SPION-labeled stem cells for the diseases related to brain aging cited. No reports had severe adverse effects (death, disability, etc). An improvement in the immunoregulatory condition within 24 hours after the MSC implantation was also observed.

Imaging techniques to assess the therapy efficacy
The SC/SPION behavior or scattering in the nervous system as well as their habilitation or functional improvement after implantation can be performed by a technological arsenal. This arsenal varies from technical or histological tests (Berlin blue, Prussian blue, cresyl violet, etc), immunohistological (antibody to tyrosine hydroxylase, glial fibrillary acidic protein, etc), to molecular (real-time polymerase chain reaction [rtPCR]), Bioplex assay to check the immunological status, etc) in ex vivo tissue or post mortem, see Table 6.
There are tests that can be performed in vivo, such as functional recovery by radioisotopes (PET or SPECT) or complexed fluorophores with substances that mimic the neurochemical of neural systems or networks. Briefly, these articles addressed several neuroimaging techniques (Table 6) using in vivo and ex vivo techniques. All studies used MRI and various magnetic fields from 1.5 tesla (T) 13,24,25,70,72 to 9.4 T. 14,21,33,67,69,73 The next paragraphs describe the use of these technologies in each neurodegenerative disease.

Stroke
MRI equipment was used in all stroke studies to monitor SC/SPION distribution in vivo (Table 6). MRI used magnetic fields that ranged from 1.5 T 13,70,72 to 9.4 T 14,67,69,73 and the sequence most used was 3D spin echo, weighting T2. The average monitoring period was 13 days, ranging from the day of SC/SPION injection to 70 days 68 after implantation. The ex-vivo analysis was observed in most studies, and they generally used the immunohistochemistry Prussian Blue assay to locate SC/SPION after implantation in several brain and/or peripheral regions, as described in the previous section.
In most studies we observed the concomitant presence of iron and stem cells in the ischemic region affected by stroke. [12][13][14]63,66,67,69,71,73,76 In addition, the presence or over expression of a series of molecules in the ischemic region was also observed by immunohistochemistry. The most prevalent was the increased expression of GFAP, followed by NeuN, vimentin, and tubulin, suggesting that stem cells' presence can change the ischemic microenvironment and promote structural support reorganization, enabling proliferation of new cells. 12,67,68,70,71,73,74,76 Imaging methods most frequently used in these studies were flow cytometry 12,14,62 followed by microscopy, confocal microscopy, 71 electronic microscopy, 67 electrophysiology of potassium channel, 12 or microarray/cytogenetic. 13,14 Parkinson's disease All selected studies performed "homing" or cell migration analysis in vivo by MRI. The magnetic field of the equipment ranged from 1.5 T 24,25 to 9.4 T, 21 and most studies used the fast low-angle shot (FLASH) protocol in T2-weighted sequence, in the period ranging from 1 day 22,24 to 24 weeks, or 168 days 23 after implantation of SC/SPION. Two studies 23,24 reported the presence of hypo signal in the striatum and substantia nigra. 23,24 All studies that used histological techniques to analyze the SC/SPION therapeutic efficacy ex vivo perfused animals with 4% paraformaldehyde and conducted an immunohistochemical assay for tyrosine hydroxylase (TH), reporting an increased number of TH positive neurons. Other studies 20,21,25 observed an increase in signaling of neural growth factors, and neuroprotective proteins such as GFAP after 6 weeks. 21 This dopaminergic recovery was also observed in PET, 21 22 and more recently in rtPCR 24,25 (Table 4).

Multiple sclerosis and ALS
All studies used MRI technique to monitor SC/SPION distribution through the brain after intrathecal 34 or intracerebroventricular 30-33 cell application ( Table 5). The magnetic fields ranged from 1.5 T 34 to 9.4 T, 33 the spin echo sequence 31,32 was more frequent, and the mean monitoring period was 32 days, ranging from 24 hours after implantation 30,31,34 to 18 weeks. 32 One study 30 used Prussian Blue for ex vivo analysis, the other studies used immunohistochemistry assays for class I -human leucocyte antigens 31 and GFAP. 32,33 All these markers were enhanced in the regions of motor tracts of the brain after 7 days of SC/SPION administration. Two studies 32,34 used Bioplex equipment to check the immunephenotypic condition, and they also observed a significant improvement even after the period previously reported. In

Discussion
The brain aging process is characterized by a reduced cortical area, and reduction in the number of neurons and their connections. 78 This process is multifactorial and the individual's lifestyle is considered to be a high impact factor in the aging process. 79,80 Obesity, stress, and depression accelerate this process. 80 A nutritional diet with polyphenolic compounds, curcumin or glucose restriction, physical activity, and mentally stimulating pursuits are considered beneficial factors. 81 Genetics, in addition to lifestyle also play central roles in this process. 82 There are several studies suggesting an increased expression in miRNA network that modulates the inflammatory profile of neural and glial cells, also affecting behavioral aspects and, therefore, influences the epigenetics, 79,83 or the cells' telomerase activity by accelerating or slowing down the process of cerebral aging. 84 In this sense, accelerated brain aging by environmental components, behavioral or genetic, makes the brain structure vulnerable to two distinct disease processes, which are associated in some situations: i) oxidative stress modulating the chronic accumulation of protein clusters (a-synuclein, β amyloid, Tau, etc) or intracellular apoptotic/degenerative events by the increased immune response, and ii) the ischemic event, acute in most situations, caused by trauma or thromboembolic events. 48 AD, Parkinson's disease, stroke, and sclerosis are the most prevalent diseases related to the cerebral aging process. 54 Cell therapy is a nonpharmacological option to treat the mentioned diseases. 48 This technique sometimes restores neural population through a process of cell transdiffere ntiation, 51,85 growth factor releasing and/or modulating the inflammatory microenvironment, 54,86 and is therefore emerging as a therapeutic option in diseases refractory to other treatments. The MSC are multipotent, 87 and besides providing the beneficial aspects described above, they have greater safety regarding teratogenicity; 88,89 however, these cells can migrate randomly, mainly to inflammatory or apoptotic sites. 87 For this reason, the goal of this review was to systematically analyze scientific data of pre-clinical and clinical trials to provide an overview of the use of SPION labeled stem cells for diseases related to the aging brain process, such as ischemic stroke, Parkinson's disease, sclerosis, and dementia/AD.
The use of SPION labeled stem cells for cell therapy for diseases related to cerebral aging is not new. The first study 90 on this subject dates back to 1988. The majority of studies analyzed in this review used commercial compounds, especially Feridex ® (Endoren ® ) adopted by 18 studies of the 32 included (57%), and 24 of 32 included (75%) studies adopted dextran coated compounds. Although literature abounds with studies on the use of SPION for labeling MSC, this technique was questioned by Guenoun et al 91 after this author established that paramagnetic gadolinium contrast enabled a better internalization efficiency and higher cell viability by the use of trypan blue. However, Jasmin et al 92 remarked that induction of reactive oxygen species by this contrast can affect the cells' proliferative potential. In our review, the main transfection agent identified in 12 of 32 (38%) selected articles was PLL. Studies 93,94 suggest that PLL use facilitates the SPION internalization process by MSC in MRI analysis, decreasing the toxicity related to SPION concentration, to the period of stem cell and SPION incubation.
DMEM was the main culture medium used (26 of 32 [86%]), and all aforementioned studies also used DMEM for SPION or gadolinium internalization with stem cells. Accordingly, the SPION and stem cells' incubation period ranged from 1 to 72 hours, and most (12 of 32 [32%]) studies used 24 hours. The Jasmin et al 92 findings corroborate this 3767 SPION-labeled stem cells for the diseases related to brain aging fact by showing that SPION MRI is significantly improved after 24 hours, 92 and that cell viability is reduced after 30 hours. This same finding was observed in the Mamani et al study. 93 Only two publications used multimodal nanoparticles 14,72 for stem cell labeling, and one of them used the complexation technique with fluorgold. Another study used a commercial compound named FluidMag. The use of multi-or polymodal particles is new in the study of stem cell therapy functionality. 92 The most commonly used stem cells in the selected articles were MSC (65%), followed by embryonic stem (15%), neural stem cell (12%), hematopoietic stem cell (4%), and stem cells from amniotic fluid (4%). The same distribution of the stem cells used was also observed in a review published in Cell Transplantation, between 2008 and 2009. 95 The origins or sources of stem cells used in publications were human (118 of 214 [55%]), followed by mouse (49 of 214 [18%]). These results corroborate our analysis that identified eleven studies (42%) using human stem cells and eight studies (38%) using rat cells. In this sense, most studies used heterologous cells, since 98% of studies used rodents. In addition, we also observed the use of immunosuppressive drugs such as cyclosporine, in ten of eleven studies of heterologous therapy.
The SPION labeled stem cell therapy detected more evident in upper magnetic fields, but in all MRI fields identified (1.5 to 11T) and protocols (Table 6), the SC/ SPION were location in the therapeutic target, penumbra region for stroke, nigrostriatal region for Parkinson's disease, and regions of the internal capsule and corticospinal tract for ALS. This result was also evidenced by our group and by other articles. 48,88,89,95 The functional improvement was identified by an extensive variation in the method of measurement among selected groups of diseases (stroke, Parkinson's disease, sclerosis).
Among the 20 selected studies on stroke, only one studied 65 the effects of stem cell therapy in patients up to 30 days after the ischemic event, and found that therapy efficacy was low in several functional outcomes analyzed (cognition, motor skills, socio-familial, etc). Recent experimental studies showed that MSC are more efficient at modulating the neural microenvironment by promoting growth factor release and glial activation, mainly of oligodendrocytes, which enables better structural and functional reorganization compared with hematopoietic stem cells used in this study. 96,97 This observation is supported by articles included in this review, which, besides confirming the ischemic region reduction after 7 days after stem cells' administration in both photothrombosis and temporary occlusion of the middle cerebral artery model, also showed an improvement in memory behavioral tests, such as the Morris water maze, and adapted neurological scales. For the stroke model most of the articles (13 of 32 [65%]) used the middle cerebral artery occlusion, and others conducted a temporary occlusion of 1 hour on average. Two studies underwent permanent occlusion as control injury. Some reviews in the literature highlight the model by temporary occlusion of the middle cerebral artery as the most widely used model, because there is the advantage in not performing a craniotomy, and restriction of the affected area overcomes the disadvantage of the injury not being generated by a thromboembolic event. 98,99 The use of molecular biology techniques has increased (PCR, Western blot, ELISA, etc). In articles selected for this review the functional genomics in experimental studies of stroke was observed in microarray 12,13 and cytogenetics. 14 The model for Parkinson's disease induction was neurotoxin 6-OHDA injected at the MFB. The articles reported functional improvement using the apomorphine rotation test, on average, after 3 weeks. It was also observed that animals undergoing cyclosporine therapy before receiving stem cells in the striatal region, had functionally recovered after 7.3 weeks. This result was demonstrated through rotational test using methamphetamine and neuroimaging exams. MRI presented magnetic fields ranging from 1.5 to 9.4 T, and the protocol FLASH parameter-weighting T2 was the most frequent.
In general, after 11.3 weeks animals were sacrificed and perfused with 4% paraformaldehyde. The animals' brains were removed and mainly the CPU/striatum and substantia nigra were examined by immunohistochemical assay, and found increased tyrosine hydroxylase and TH positive neurons. This dopaminergic recovery was also observed by other methods, such as PET (Bjorklund et al 19 ; Jackson et al 21 ), HPLC, 22 and more recently by rtPCR. 15,24 The articles that evaluate the use of SPION labeled stem cells in MS identified that the SPION internalization process did not modify the stem cells' viability, proliferation, metabolism, and phenotype. 32 Furthermore, it seems a good therapy for ALS, 29,31,32 since the motor skills significantly improve in behavioral tests and electrophysiology about 7 days after stem cells' implantation. Therefore, there is extensive literature on cell therapy benefits for ALS, 100 MS, 101 sclerosis, and other demyelinating diseases.
According to the presented data, nanobiotechnology constitutes a powerful tool to study stem cells' use to treat International Journal of Nanomedicine 2014:9 submit your manuscript | www.dovepress.com

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Alvarim et al diseases related to the aging brain, particularly for allowing tracking of these cells in vivo. Due to the central nervous system complexity, cell-based therapy is a challenge, and nanomedicine tools for monitoring and guiding these studies are highly desirable.