Regenerative Medicine for Cerebral Infarction

Tissue plasminogen activator (t-PA) is the gold standard drug for cerebral infarction in the acute phase (Adams et al., 2007), but it cannot be administered to all cerebral infarction patients. Some patients who survive the acute phase of cerebral infarction suffer from permanent hemiparesis in the chronic phase, which highlights the need for regenerative medicine to play a more important role in treating such individuals. There are two primary approaches to the use of regenerative medicine for patients with cerebral infarction: exogenous stem cell therapies, and enhancement of endogenous stem cells. Stem cell transplantation is one of the most widely employed strategies using exogenous stem cells. Many studies with experimental animals have shown that stem cell transplantation enhances functional recovery after cerebral infarction (Kameda et al., 2007; Takahashi et al., 2008). Based on the results of animal experiments, several clinical trials for patients with cerebral infarction are currently ongoing, using stem cell transplantation techniques, typically with mesenchymal stem cells (Detante et al.). However, these clinical trials have been started despite a lack of results showing that transplanted stem cells can reliably replace infarct areas. The principal purpose of cell transplantation in these cases is cell replacement, or replacement and restoration of infarct areas. Nevertheless, only a few percent of the transplanted cells typically survive during the chronic phase of cerebral infarction (Lindvall & Kokaia, 2006). Even more problematic is the fact that few of these transplanted stem cells differentiate into neurons with immunohistological and electrophysiological properties (Anderova et al., 2006). Based on these reports, some scientists maintain that functional improvements can be achieved without cell-replacement, that the effects of trophic factors secreted by the transplanted cells are sufficient (Cabrer et al., 2010; Shimada & Spees, 2011). Another approach that regenerative medicine can take is enhancement of endogenous stem cells, based on methods that are less invasive than the use of exogenous stem cells. Deep brain stimulation (DBS) for Parkinson’s disease patients is an example of a standard therapy now used in clinical situations. A previous report using animal subjects has shown that DBS can enhance the neurogenesis of endogenous stem cells (Toda et al., 2008). Based on this report, we evaluated the effectiveness of electrical stimulation on animals with cerebral infarction. Recently, we showed that electrical stimulation of the cerebral cortex during the acute phase of cerebral infarction exerted anti-apoptotic, angiogenic and anti-inflammatory effects through the PI3K-Akt signaling pathway (Baba et al., 2009). Moreover, we showed


Introduction
Tissue plasminogen activator (t-PA) is the gold standard drug for cerebral infarction in the acute phase (Adams et al., 2007), but it cannot be administered to all cerebral infarction patients.Some patients who survive the acute phase of cerebral infarction suffer from permanent hemiparesis in the chronic phase, which highlights the need for regenerative medicine to play a more important role in treating such individuals.There are two primary approaches to the use of regenerative medicine for patients with cerebral infarction: exogenous stem cell therapies, and enhancement of endogenous stem cells.Stem cell transplantation is one of the most widely employed strategies using exogenous stem cells.Many studies with experimental animals have shown that stem cell transplantation enhances functional recovery after cerebral infarction (Kameda et al., 2007;Takahashi et al., 2008).Based on the results of animal experiments, several clinical trials for patients with cerebral infarction are currently ongoing, using stem cell transplantation techniques, typically with mesenchymal stem cells (Detante et al.).However, these clinical trials have been started despite a lack of results showing that transplanted stem cells can reliably replace infarct areas.The principal purpose of cell transplantation in these cases is cell replacement, or replacement and restoration of infarct areas.Nevertheless, only a few percent of the transplanted cells typically survive during the chronic phase of cerebral infarction (Lindvall & Kokaia, 2006).Even more problematic is the fact that few of these transplanted stem cells differentiate into neurons with immunohistological and electrophysiological properties (Anderova et al., 2006).Based on these reports, some scientists maintain that functional improvements can be achieved without cell-replacement, that the effects of trophic factors secreted by the transplanted cells are sufficient (Cabrer et al., 2010;Shimada & Spees, 2011).Another approach that regenerative medicine can take is enhancement of endogenous stem cells, based on methods that are less invasive than the use of exogenous stem cells.Deep brain stimulation (DBS) for Parkinson's disease patients is an example of a standard therapy now used in clinical situations.A previous report using animal subjects has shown that DBS can enhance the neurogenesis of endogenous stem cells (Toda et al., 2008).Based on this report, we evaluated the effectiveness of electrical stimulation on animals with cerebral infarction.Recently, we showed that electrical stimulation of the cerebral cortex during the acute phase of cerebral infarction exerted anti-apoptotic, angiogenic and anti-inflammatory effects through the PI3K-Akt signaling pathway (Baba et al., 2009).Moreover, we showed the previous results from animal experiments as well as the current clinical situation, autologous stem cell transplantation is considered to be of significant benefit to patients who were not administered rt-PA, provided it is performed in the acute phase three hours after onset.

Delivery methods
There are several stem cell delivery methods, such as intraparenchymal transplantation, intravenous administration and intraarterial administration.With any of these methods, the number of surviving transplanted donor cells is thought to affect the extent of recovery from cerebral infarction.Intraparenchymal transplantation can be performed so that stem cells are delivered in the ischemic penumbra, and it is thus the best method for placing the largest number of stem cells in the desired area of the brain, but this is the most invasive method.Intravenous administration, on the other hand, is the least invasive, but most stem cells end up in the liver and lung (Wang et al., 2010), leaving a much smaller number of stem cells surviving in the area of the ischemic penumbra compared with the intraparenchymal transplantation method.This is why the quantity of stem cells administrated intravenously in animal experiments is roughly an order of magnitude larger than that used in other delivery methods (Li et al., 2008;Lundberg et al., 2011).Lappalainen et al. detected an accumulation of graft cells which were intraarterially administrated in the ischemic brain, using SPECT/CT, but such cells were not observed when administrated intravenously (Lappalainen et al., 2008).At the present time, relatively few papers have explored intraarterial methods of administering stem cells for cerebral infarction in animal models.However, an endovascular technique, superselective intraarterial administration to the penumbra via a micro-catheter, can be performed in a clinical situation, and this method is expected to be less invasive than intraparenchymal transplantation.

Graft survival
Although the therapeutic effect of stem cell transplantation depends upon the rate of stem cell survival, research to date has reported that only approximately 5% survives after transplantation (Lindvall et al., 2004).Cytoprotection can enhance the percentage of graft survival.In particular, GDNF has been shown to be an effective neurotropic factor against ischemic injury.Its neuroprotective effect mainly derives from activation of the phosphatidylinositide-3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase/ERK (MAPK/ERK) pathways (Nicole et al., 2001;Treanor et al., 1996).During transplantation, stem cells are subject to hypoxic-ischemic injury.Wang et al. showed that graft survival was enhanced by pretreatment with GDNF for three days before NSC transplantation (Wang et al., 2011).Because brain tissue architecture is disrupted in the ischemic brain, the use of biodegradable scaffolds may help transplanted stem cells regenerate and/or restore damaged brain structures and functions, by affecting cell differentiation, morphology, adhesion, or gene expression (Kleinman & Martin, 2005;Steindler, 2002).Jin et al. showed that transplantation of human neural precursor cells (NPCs) in Matrigel scaffolding at the time of transplantation partially improved therapeutic outcome compared to that of NPCs without Matrigel scaffolding, and that the use of NPC/Matrigel cultures dramatically improved the therapeutic effect (Jin et al., 2010).These reports indicate that, when using transplantation methods employing pre-treatment with GDNF, or Matrigel scaffolding, cytoprotective effects that enhance the survival rate of stem cells require time to develop in vitro before transplantation and that such development might be related to the enhanced cytoprotective effects observed after transplantation.

Functional recovery mechanisms: cell-replacement versus paracrine effects
In experiments with animals, many researchers have confirmed that stem cell transplantation provides neuroprotective effects immediately after transplantation, based on behavioral analyses and histological analyses that show reductions in infarct volume (Kameda et al., 2007;Kurozumi et al., 2004;Takahashi et al., 2008).Histological analyses also showed that the neuroprotective effects were due to enhanced angiogenesis (Onda et al., 2008), anti-apoptotic effects (Kameda et al., 2007;Kurozumi et al., 2004), and so on.A recent paper showed that mononuclear bone marrow cells played a role in a rapidly developed neuroprotective effect by increasing cerebral blood flow six hours after transplantation, followed by evidence of angiogenesis (Fujita et al., 2010).Yilmaz et al. described remarkable induction of genes for nerve guidance survival (e.g., cytokine receptor-like factor 1, glypican 1, Dickkopf homolog2, osteopontin), as well as increased expression of neurogenerative, nerve guidance, and angiogenic factors (bFGF, bone morphogenetic protein, angiopoietins, neural growth factor), after transplantation with bone marrow stromal cells (Yilmaz et al., 2010).Angiogenesis and anti-apoptotic effects are preferable for neuroprotection, and if the goal were limited to functional recovery, these neuroprotective mechanisms might be sufficient.However, in a strict sense, stem cell transplantation is expected to provide for cell replacement, since stem cells have two outstanding capacities, namely, self-renewal and pluripotency, which means that they can produce neurons, astrocytes and oligodendrocytes (Gage, 2000;Okano, 2002;Temple, 2001).Cell-replacement therapy requires that transplanted stem cells survive in the damaged brain, differentiate into mature cells, then replace neurons of several phenotypes, and reconstruct new networks with host cells.Several approaches have been studied to enhance neuronal differentiation, and one approach is to transplant site-specific cells.If the sitespecific characteristics of NSCs can be maintained during in vitro expansion, such cells may differentiate into site-specific neurons after transplantation.Another approach is to modify the cellular characteristics of the stem cells differentiation by transfecting a trophic factor gene (Kameda et al., 2007;Kurozumi et al., 2004).We have showed that, compared with unmodified stem cells, neuronal differentiation is enhanced by transplanting into the ischemic brain adult neural stem/progenitor cells that were modified to secrete GDNF.This enhancement of the differentiation is usually difficult to detect in the ischemic core, and is typically found only in the small ischemic boundary zone.Also, we are still not able to effect a complete replacement of the damaged infarct area using transplanted stem cells.Liu et al. have shown that DCX-expressing immature neurons in the subventricular zone (SVZ) do not exhibit a Na+ current, and their resting membrane potential is approximately -25mV in the absence of ischemic insult, however, after ischemic insult, such neurons do exhibit a Na+ current, and the membrane potential is hyperpolarized to about -54mV, a voltage similar to that of mature neurons.They also showed that gene analysis indicates that immature DCX cells express immature markers for Sox 2 and nestin in the absence of ischemic insult, but tyrosine hydroxylase (TH) is expressed as a mature marker after ischemic insult (Liu et al., 2009).After ischemic insult, immature stem cells become able to express the same phenotypes as mature neurons.
Research published during the last three years, however, indicates that cell replacement via stem cells transplantation is not essential to functional recovery (Ramos-Cabrer et al., 2010;Shimada & Spees, 2011).Previously, there was a research trend that aimed specifically at developing cell replacement therapies, as many researchers sought better methods to improve graft survival rates and enhance neuronal differentiation.Nevertheless, the present survival rate for transplanted cells during the chronic phase of cerebral infarction remains in the single digits (Lindvall et al., 2004), and few transplanted stem cells differentiate into neurons with immunohistological and electrophysiological properties (Anderova et al., 2006).This has been interpreted by some researchers as indicating that functional improvements can be achieved without cell replacement, and that the effects of trophic factors secreted by the transplanted cells are sufficient.Ramos-Cabrer et al. recently have shown that post-stroke functional recovery after stem cell transplantation is due to paracrine mechanisms, not cell replacement.They found no evidence of surviving grafted stem cells six months after stem cell transplantation, and, compared with control animals, functional recovery was confirmed even during the chronic phase of cerebral infarction (Ramos-Cabrer et al., 2010).This report supports a new interpretation concerning the importance of paracrine mechanisms to functional recovery from cerebral infarction.Based on this new interpretation, clinical trials using stem cells to treat cerebral infarction are presently ongoing (Detante et al.).

Future directions
An increasing number of research papers on stem cell transplantation are focused on the neuroprotective effects provided by paracrine mechanisms (Shimada & Spees, 2011;Sun et al., 2010).Although cell replacement therapy is likely to remain an important target of stem cell transplantation, the focus on paracrine mechanisms will spur the development of clinical trials, for which long-term efficacy and safety are crucial evaluative factors.Thus, standardization of techniques will be more important compared with the procedures used in animal experiments.Furthermore, it has been reported that the quality of transplanted stem cells profoundly affects the functional outcome.Assmus et al. reported the results of the REPAIR-AMI (Reinfusion of Enriched Progenitor cells And Infarct Remodeling in Acute Myocardial Infarction) trial, and showed that contamination by red blood cells affected the functionality of isolated bone marrow-derived progenitor cells, and ultimately inhibited recovery from acute myocardial infarction (Assmus et al., 2010).An increasing number of clinical trials applying stem cell therapies for cerebral infarction will be started in the near future, and cell preparation will play a vital role for proper interpretation of the results.

Enhancement of endogenous stem cells 2.2.1 Long-term potentiation (LTP)
Another useful approach for regenerative medicine in cerebral infarction cases is to enhance endogenous stem cells.LTP is thought to be a cellular and molecular mechanism of hippocampal learning and memory (Bliss & Collingridge, 1993).LTP is observed as a longlasting enhancement in the efficacy of synaptic transmissions, which requires NMDA receptor activation and increased Ca 2+ influx.LTP can be induced by brief high-frequency stimulation.Chen et al. showed that high-frequency stimulation or tetanic stimulation induced the release of Wnt3a from hippocampal neurons (Chen et al., 2006).Wnt3a has been shown to be a major regulator of neurogenesis in vivo and in vitro, and blocking Wnt3a expression has been reported to cause a significant decline in neuronal generation (Davidson et al., 2007).Moreover, recent papers suggest that LTP per se enhances neurogenesis (Bruel-Jungerman et al., 2006;Chun et al., 2006Chun et al., , 2009)).These findings, linking LTP stimulation to the activation of a large latent neural precursor pool in the dentate gyrus, could explain the ability of specific environmental stimuli to increase the rate of neurogenesis in the hippocampus over prolonged periods.

Exercise
It is now widely accepted that exercise (van Praag et al., 1999), enriched environments (Kempermann et al., 1997), and learning tasks (Gould et al., 1999), can enhance the neurogenesis of endogenous stem cells, and affect regulatory mechanisms that may be linked to LTP.Exercise has been shown to enhance neurogenesis in both intact and disease animal models.Tajiri et al. showed that exercise had neuroprotective effects on a Parkinson's disease model in rats, with enhanced neurogenesis and migration toward the lesioned striatum observed.Furthermore, brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) showed increases in the striatum as a consequence of exercise (Tajiri et al., 2010).Li et al. showed that whisker stimulation, as a peripheral stimulation after focal barrel cortex ischemia, enhanced migration from the subventricular zone, and increased the neurogenesis of endogenous stem cells, due to increased vascular endothelial growth factor (VEGF) and stromal-derived factor-1 expression (SDF-1) in the penumbra.They also showed that local cerebral blood flow recovered to a greater degree due to whisker stimulation (Li et al., 2008).This indicates that increased peripheral stimulation and afferent signals to the ischemic cortex can activate endogenous neural stem cells, cause them to migrate to the injured region, and differentiate into mature neurons.Thus, the beneficial aspects of rehabilitation are recognized as being vital to long-term recovery from ischemic stroke.

Electrical stimulation and repetitive transcranial magnetic stimulation
Electrical stimulation and repetitive transcranial magnetic stimulation have been used in clinical situations for treatment of many central nervous system diseases, for example Parkinson's disease (PD) (Fasano et al., 2010), epilepsy (Fisher et al., 2010), depression (Horvath et al.,2010) , and chronic pain (Rasche et al., 2006).Deep brain stimulation (DBS) has become a standard clinical therapy for PD patients and it can ameliorate motor function in such individuals, although the mechanism remains poorly understood.Toda et al. showed that DBS of the anterior nucleus of the thalamus (AN) enhanced the presence of endogenous stem cells in the hippocampal dentate gyrus and enhanced neurogenesis, which were associated with enhanced behavioral performance.Moreover, they showed that DBS of the AN reversed steroid-induced reductions in neurogenesis (Toda et al., 2008), implying that DBS can modulate synaptic plasticity and hippocampal neurogenesis.Based on these reports, we have tried to evaluate whether electrical stimulation has a neuroprotective effect that mitigates cerebral infarction damage by the enhancement of endogenous stem cells and neurogenesis.We began with epidural stimulation because we wanted to stimulate the brain using a method that is less invasive compared with the use of exogenous stem cells for stem cell transplantation.In one of our experiments, rats received continuous electrical stimulation above the cerebral cortex during the acute phase of cerebral infarction.This stimulation increased cerebral blood flow, enhanced behavioral recovery and reduced infarct volume.The neuroprotective effect was derived from antiapoptotic, angiogenic and anti-inflammatory effects through the PI3K-Akt signaling pathway (Baba et al., 2009).As a next step, we hypothesized that electrical stimulation of the striatum could enhance the proliferation, migration, and neuronal differentiation of endogenous stem cells in the subventricular zone even during the chronic phase of the ischemic brain.Striatal electrical stimulation during the chronic phase of cerebral infarction was observed to enhance behavioral recovery and reduce infarct size.This neuroprotective effect was derived from stimulation of endogenous angiogenesis and neurogenesis with GDNF and VEGF upregulation (Morimoto et al., 2010).Machado et al. showed that chronic contralesional electrical stimulation of the lateral cerebellar nucleus improved motor recovery in rats following ischemic strokes, an effect derived from increased perilesional cortical excitability via chronic activation of the dentatothalamocortical pathway (Machado et al., 2009).Transcranial direct current stimulation (tDCS) has been used in animal experiments and for chronic stroke patients.tDCS is thought to strengthen synaptic connections (Cheeran et al., 2008;Hummel et al., 2005;Nitsche et al., 2003Nitsche et al., , 2004)), through a mechanism similar to that of LTP.Fritsch et al. showed that tDCS improved motor skill learning through enhanced synaptic plasticity that required brain-derived neurotrophic factor (BDNF) secretion and TrkB activation (Fritsch et al., 2010).Vagus nerve stimulation (VNS) has been used in an animal model of ischemia, and patients given this therapy have demonstrated enhanced behavioral recovery and reduced infarction size (Ay et al., 2009).The potential mechanisms for the observed beneficial effects of VNS are thought to be the suppression of increased neuronal excitability, and the reduction of cytokine overproduction and inflammation.Collectively, electrical stimulation has been shown to have therapeutic benefit in cases of cerebral infarction not only in the acute phase but also in the chronic phase, which suggests that electrical stimulation has considerable therapeutic potential.In addition to electrical stimulation, repetitive transcranial magnetic stimulation (rTMS) has been used in animal models of ischemia (Kaga et al., 2003), and in infarct patients with aphasia (Weiduschat et al., 2011), and has shown enhanced functional recovery.Compared with electrical stimulation, rTMS is more widely used because patients can avoid the surgery required for electrode implantation.It is presumed that the mechanism of its neuroprotective effect derives from increased glucose metabolism, inhibition of apoptosis in the ischemic hemisphere (Gao et al., 2010), and increased expression of c-fos, which is followed by upregulation of BDNF (Zhang et al., 2007).

Rehabilitation combined with electrical stimulation
Currently, after cerebral infarction, electrical stimulation (especially epidural electrical stimulation) is mainly performed together with rehabilitation, to enhance the functional recovery that normally occurs during rehabilitation.Northstar Neuroscience has performed clinical trials using epidural electrical stimulation for infarct patients with upper extremity hemiparesis (Northstar Neuroscience, formerly of Seattle, WA, U.S.A.).Unfortunately, the results of a Phase III randomized trial were unsuccessful, but the Phase II study showed that this therapeutic intervention is both safe and effective.Recently, preliminary study results from the same group showed that patients with non-fluent aphasia benefitted from speechlanguage therapy in combination with epidural electrical stimulation of the premotor cortex, identified by fMRI.Patients with moderate as well as severe aphasia showed functional improvements after epidural electrical stimulation (Cherney et al., 2010).

Parameters and stimulation patterns
As mentioned above, electrical stimulation and transcranial magnetic stimulation can be of significant functional benefit to individuals who have suffered a cerebral infarction.However, stimulation parameters can vary widely even for the same stimulation method.F o r e x a m p l e , i n s o m e o f o u r r e s e a r c h o n e p i d u r a l s t i m u l a t i o n , w e u s e d c o n t i n u o u s stimulation with 2Hz pulses of 1ms width at an intensity of 100μA.On the other hand, Moon et al. used intermittent stimulation with 50Hz pulses of 194ms width whose intensity was flexibly adjusted to evoke movement of a forelimb.They also compared the duration of stimulation, and observed that compared to continuous stimulation, intermittent stimulation enhanced functional recovery more effectively (Moon et al., 2009).The neuroprotective and neurorestorative effects of electromagnetic stimulation depend on the stimulation parameters and pattern.Moon et al. mentioned that the pattern and intensity of stimulation should be modified on an individual basis depending on the extent of the infarct.The efficacy of the results is affected by a large number of parameters, such as the frequency, intensity, pulse width, and duration of the stimulation (i.e., whether it is continuous or intermittent), and the stimulation target area and electrode resistance.Since the best combination of stimulation parameters and pattern are unknown at the outset, researchers tend to stimulate the ischemic brain using different stimulation parameters and patterns, searching for the combination that best enhances the neuroprotective and neurorestorative effects.Regarding the frequency of stimulation, the difference in effect between high-frequency stimulation and low-frequency stimulation is usually explained as a consequence of different cellular and molecular mechanisms, LTP versus long-term depression (LTD), because synaptic plasticity is one of the mechanisms responsible for enhanced functional recovery due to electrical stimulation.Based on numerous previous reports, brief highfrequency stimulation (100Hz or higher) can induce long-term potentiation (LTP).On the other hand, brief low-frequency stimulation (1 or 2Hz) can induce long-term depression (LTD), which impairs long-lasting enhancement of synaptic transmission.Thus, the mechanism of enhanced recovery observed in response to high-frequency stimulation is discussed in terms of LTP, whereas low-frequency stimulation fails to induce functional recovery.The duration of stimulation also affects the outcome.Brief high-frequency stimulation induces LTP, but this does not mean that continuous high-frequency stimulation will do the same.In a preliminary study, we could not confirm reductions in infarction volume after continuous high-frequency stimulation, compared with application of low-frequency stimulation (Baba et al., 2009).This implies that inappropriate parameters and stimulation patterns may simply cause tissue damage, and provide no therapeutic effect.The condition of the brain also affects the results.Most LTP experiments conducted in the field of electrophysiological research are performed using intact rats and mice.As described above, NMDA receptor activation and increased Ca 2+ influx are required for the induction of LTP.Increased Ca 2+ influx is also observed in ischemia and because the ischemic brain has already been exposed to increased Ca 2+ influx, the response to high-frequency www.intechopen.comstimulation that can induce LTP in intact animals would be different in animals with cerebral infarction.Although LTP is a possible mechanism whereby electrical stimulation enhances functional recovery, it is likely that other mechanisms are also involved.Electrical stimulation can induce increased regional cerebral blood flow, suppress inflammatory responses, induce anti-apoptotic responses, and enhance angiogenesis, which, individually and in combination, modulate the microenvironment of the infarct brain to enable functional recovery.This implies that electrical stimulation should be performed using appropriate parameters and stimulation patterns that are tailored for the condition of the brain.

Future directions
Due to a recent finding, that aged mice contain a larger pool of latent stem cells than can be activated (Walker et al., 2008), determining the best parameter settings and pattern of electromagnetic stimulation that will yield the best possible functional outcomes in patients with cerebral infarction is of paramount importance.To find ideal parameter values and patterns, the mechanism of electrical and magnetic stimulation must be elucidated in more detail.Given the failure of the Northstar Neuroscience Phase III trial in which epidural electric stimulation was used, rTMS will likely play a more important role for cerebral infarction patients in the future, because it is a less invasive technique.And, although improving cerebral infarction treatment procedures is of vital importance, primary stroke prevention is also essential.Simvastatin enhances hippocampal LTP in mice and causes a significant increase in Akt phosphorylation (Mans et al., 2010), and since LTP per se can enhance neurogenesis, as mentioned earlier, this medication should be effective for preventing primary stroke as well as hyperlipidemia.In short, the protection and enhancement of endogenous stem cells may be a key factor in the maintenance and prolongation of health.

Progress in ischemia analysis methods
Thus far, we have outlined basic therapeutic strategies for treating cerebral ischemia, based on the use of exogenous stem cells and the enhancement of endogenous stem cells.Improvement in therapeutic strategies and further elucidation of the ischemia mechanism in greater detail are both important.To elucidate the mechanism of ischemia, and the actions of regenerative medicine, we need to analyze the neuronal activity in the ischemic brain by examining the response of single cells, as well as the response of large populations of neurons.For this purpose, the combination of MRI examinations (especially fMRI) and electrophysiological analysis are useful, because fMRI signals are thought to be proportional to the local average of neuronal activity.The use of fMRI provides a major breakthrough not only in animal experiments but also in treatment of patients.As mentioned above, fMRI enables the enhancement of patient rehabilitation after stroke, via epidural electrical stimulation and detection of premotor cortex function.Using fMRI techniques, functional recovery after ischemia can be monitored, as originally functional areas are reactivated, with preservation of neurovascular coupling (Weber et al., 2008).fMRI also can reveal ipsilateral cortical fMRI responses after peripheral nerve damage, so that increased interneuron activity can be observed.Thus, fMRI enables analysis of modifications in fiber connections, such as callosal interhemispheric projections (Pelled et al., 2009).The use of voltage-sensitive dyes also provides a similar correlation with extracellular direct current potential recording, which enables the analysis of molecular mechanisms of ischemia from an electrophysiological point of view (Farkas et al., 2008).Voltage-sensitive dye techniques allow sensory-evoked depolarization after cerebral infarction to be analyzed in considerable detail (Siglera et al., 2009).The comparison of results derived from MRI examinations with those obtained from electrophysiological analysis would also be useful.The ischemic penumbra is a major target when attempting to treat cerebral infarction.From an electrophysiological point of view, depolarization is induced in the ischemic core and brief depolarization is induced in periinfarct areas.Unlike electrophysiological analysis, diffusion-weighted imaging (DWI) in MRI does not include peri-infarct areas, defined as areas where a brief depolarization is seen during an electrophysiological examination.Thus, electrophysiological analysis can detect wider areas of damage than those detectable using histological or MRI techniques (Breschi et al., 2010).
Behavioral analysis and fMRI analysis are typically usually used when analyzing and evaluating functional recovery, but electrophysiological analysis is seldom performed.The pursuit of cross-sectional analysis in greater depth should help to clarify the mechanism of cerebral infarction.

Conclusion
We have reviewed the therapeutic effect of stem cell transplantation and techniques for the enhancement of endogenous stem cells.As described above, previous studies have shown that functional recovery after cerebral infarction can be enhanced by stem cell transplantation, and that electromagnetic stimulation can provide neuroprotective and/or neurorestorative effects in animal models of ischemia.These results will stimulate additional clinical studies, but the development of more effective and reliable therapies will require further analysis.