Cell Therapy for Neonatal Hypoxic–Ischemic Encephalopathy

Introduction

Neonatal encephalopathy is a clinical syndrome characterized by the presence of neurological symptoms, such as a depressed level of consciousness, hypotonia, and difficulty with initiating and maintaining respiration, in the first days of life in the full-term infant. The severity of neonatal encephalopathy can be graded as mild, moderate, or severe according to the classification of Sarnat and Sarnat [1]. The proportion of severe neurological deficits in these children (mental retardation, cerebral palsy, and motor deficits) is 0% for stage 1, 30%–50% for moderate (stage 2), and >90% for severe encephalopathy (stage 3) [2].

Among other possible causes, perinatal asphyxia is the most common and most studied cause of neonatal encephalopathy and will be the focus of this review. In this regard, it has been reported that intrapartum hypoxia was present in 29% of the cases of moderate to severe neonatal encephalopathy [3].

The incidence of hypoxic–ischemic encephalopathy (HIE) is 2.5 of 1,000 live births [4]. HIE was present in >40% of the newborns with >1,500 g birth weight that died in Canadian neonatal intensive care units between 1996 and 1997 [5]. Interestingly, the percentage of neonatal deaths due to perinatal asphyxia was almost the same in developed and developing countries [6].

Although it is one of the most common causes of death and long-term neurological impairment in full-term neonates worldwide, up to now, treatment for HIE is mainly supportive.

The potential use of stem cells to reduce brain damage or to promote regeneration is a possibility that has been tested in different central nervous system (CNS) disorders. The use of stem cells needs to be investigated in the available animal models of hypoxic–ischemic neonatal encephalopathy.

In this review, we summarize previously reported studies that evaluated the potential use of stem cells to regenerate or reduce brain damage in animal models of neonatal hypoxia–ischemia (HI) (Table 1). We also discuss studies that investigated the response of the endogenous stem cell population of the brain after HI.

Fetal Neocortical Grafts

Historically, the first cell transplantation study in a model of neonatal HI used fetal neocortical grafts from embryonic day 13 rats, 1 week after HI. The transplants were well developed in 16 of 26 surviving animals, even in some animals with severe infarctions, but the cortical grafts did not have a normal lamination and were clearly distinguished from the host cortex. Also, the transplantation did not affect the extent of brain damage, comparing animals with well-developed grafts with animals with no or poorly developed grafts [7]. However, another study reported that transplantation of cell suspensions of the sensorimotor cortical region of embryonic day 16 rats, 3 days after HI, improved the motor performance of the treated animals in 2 different tests [8]. Although the use of fetal tissue for transplantation raises ethical concerns and problems related to the availability of donors in a clinical setting, these reports were the first to show that the hypoxic–ischemic brain can support the survival of transplanted cells and grafts, at least in the animal models.

Neural Stem/Progenitor Cell Transplantation

Neural stem/progenitor cells (NSCs) are cells with a self-renewing capacity, and have the potential to generate cells of both glial and neuronal lineages. NSCs can be isolated from different regions at different times during brain development, and also from the adult brain.

In vitro, NSCs are maintained in an undifferentiated state and proliferate in the presence of epidermal growth factor (EGF) and/or basic fibroblast growth factor (bFGF) to form clusters of cells, called neurospheres. These clusters are formed by a mix of multipotent neural stem cells and committed progenitors and these cells can be induced to differentiate into neurons, oligodendrocytes (OLs), and astrocytes. NSCs may also have some plasticity, giving rise to endothelial cells that have the capacity to form capillary networks [9].

Most of the studies transplanting NSCs in the hypoxic–ischemic brain evaluated the migration and differentiation potential of these cells. In one study, the so-called multipotent astrocytic stem cells (MASCs) obtained from the subventricular zone (SVZ) of newborn mice (postnatal days 1–6) were shown to migrate with no directional preference and to differentiate only in cells that resembled astrocytes, when injected into sham-operated pups. However, in the brains of hypoxic–ischemic rats, these cells migrated preferentially to the area of injury, and some of them expressed neuronal markers 14–21 days after transplantation, showing that the HI brain attracts these cells and induces neuronal differentiation [10].

In more severe cases of HI, a cavity (porencephalic cyst) corresponding to a region of lost tissue that does not support the survival of new cells is often present in the brain. One strategy to overcome this limitation in the survival ability of the donor cells is the transplantation of NSCs supported by scaffolds. When NSCs were seeded in a polyglycolic acid scaffold and transplanted into the cyst of HI animals, there was evidence of neuronal differentiation and formation of neuronal connections between the grafted cells and the host neurons [11].

In another elegant study from the same group, human NSCs (hNSCs) were transplanted into the left intracerebroventricular space 3 days after an HI injury in the right hemisphere of P7 mice. The cells migrated across the corpus callosum to areas with increased stromal cell-derived factor 1α (SDF-1α) expression in the injured cortex of the contralateral hemisphere. In vitro, treatment with the chemokine SDF-1α resulted in increased proliferation and migration of hNSCs. Also, these cells expressed CXC chemokine receptor 4 (CXCR4), the receptor for SDF-1, suggesting a possible role of this chemokine in the homing of NSCs to the damaged brain areas [12].

In addition, NSCs migrate to the injured brain even when they have already been integrated in the host brain for at least 1 week. When NSCs were transplanted into the left hemisphere of postnatal day 0 (P0) rats and the right hemisphere was left intact at P7, the cells integrated throughout the parenchyma of the left hemisphere but did not migrate to the contralateral side. However, if the right hemisphere was injured by a HI insult at P7, the cells migrated across the corpus callosum to the damaged area in the contralateral hemisphere [13].

Regarding the differentiation potential of NSCs, when the cells were injected into the infarcted region differentiation into neurons and OLs was observed, but neuronal differentiation was not seen if the cells were injected into the intact cortex [13].

Since the number of newly formed neurons in these studies was very low (around 5% of the transplanted cells differentiated into neurons), the same group genetically modified NSCs to express the neurotrophic factor neurotrophin-3 (NT-3) before transplantation, in an attempt to increase neuronal differentiation. This approach increased the proportion of NSC-derived neurons to around 20%, and >80% in the penumbra, and different subtypes of neurons were formed (GABAergic, cholinergic, and glutamatergic neurons). Also, besides the differentiation effect of NT-3 on donor cells, the possible release of this factor in the brain increased the number of neurons in a damaged region not populated by donor cells. Although it was not investigated if this effect resulted from a neuroprotective role of NT-3 or from an increase in the endogenous neurogenesis, these results suggest the potential of genetically modified NSCs to combine cell replacement and gene therapy [14].

It has also been shown that NSCs transfected to overexpress fibroblast growth factor 2 (FGF-2) proliferated and migrated more than the control NSCs when injected into brain slices or into the brain of P3 rats, but only a few neurons were formed from these cells. When transplanted into the brain of HI rats, around 50% of the cells differentiated into neurons, and the additional FGF-2 transduction increased the number of engrafted NSCs and the number of grafted cells expressing the immature neuronal marker doublecortin, compared with the non-transduced cells [15].

Taken together, these studies showed that the neonatal ischemic brain attracts NSCs and induces the differentiation of these cells toward a neuronal phenotype, although the number of neurons formed is very small. However, this process can be increased when the cells are manipulated to deliver factors that improve their migration capacity and/or the induction of a neuronal differentiation. The manipulation of NSCs is a new tool that can improve the efficiency of a stem cell therapy that aims to replace the lost neurons.

Consistent with the hypothesis that NSC transplantation may involve multiple mechanisms of action in the damaged brain instead of only replacing lost cells [16], it was reported that NSCs injected into the brain 24 h after an injury reduced the area of brain infarction of hypoxic–ischemic rats. However, this effect was only seen when chondrotinase (ChABC) was injected together with the cells. The authors suggested that ChABC could modulate the release of neurotrophic factor(s) by the NSCs and/or the sensitivity of neurons to these factors [17].

Regarding the paracrine effects, it was reported that brain-derived neurotrophic factor (BDNF) knockdown in NSCs abolished the improvements in memory induced by NSC transplantation in an animal model of Alzheimer disease. NSCs increased BDNF levels and synaptic density in the hippocampus, resulting in an improved performance in memory tasks [18].

Recently, it was shown that NSCs, when injected intravenously 72 h after reperfusion in a model of stroke, integrate the brain for at least 30 days, promoting an improvement of neurological deficits. However, most of the cells remained in an undifferentiated state, suggesting that cell replacement was not the main mechanism responsible for the improvement. Undifferentiated NSCs had an anti-inflammatory effect, reduced glial scar formation, and protected striatal neurons [19].

Subcutaneous injected NSCs may accumulate in draining lymph nodes, being in close cell-to-cell contact with dendritic cells, macrophages, and endothelial cells in an animal model of multiple sclerosis. NSCs impaired dendritic cell maturation through the release of bone morphogenetic protein-4 (BMP-4). However, clinical improvement was only seen if the transplantation was made before the disease onset. Delayed treatment did not result in any clinical improvement [20]. Human NSCs may also accumulate in the spleen when intravenously injected 2 h after a collagenase-induced intracerebral hemorrhage (ICH) in rats. If the animals were splenectomized before ICH induction, the beneficial effect of NSC treatment in the reduction of brain edema formation was lost. Also, the therapeutic effect was not observed if the cells were injected 24 h after ICH [21]. These results show that NSCs may have an immunomodulatory effect in secondary lymphoid organs when acutely injected in the blood or in the subcutaneous tissue after a CNS injury.

Although these results are promising, additional studies are needed to evaluate the mechanisms involved in the neuroprotective and regenerative potential of NSCs and to compare the use of this therapy in the acute and chronic phases of HI.

In the acute phase, it is possible that a systemic injection of NSCs could prevent neuronal death and edema formation after HIE. These cells could also exert an anti-inflammatory effect in the brain and in the periphery.

In the chronic phase, a cell replacement strategy would be necessary. However, the need for increased numbers of neurons that can differentiate into the adequate subtype (s), form new connections, and extend long axons is an obstacle that needs to be overcome. Also, HIE affects different brain areas, such as the hippocampus, thalamus, and cerebral cortex, and for this reason we speculate that different strategies may be needed to replace lost neurons in different regions. But even in the chronic phase, a paracrine effect that promotes brain plasticity and an increase in neurogenesis could result in neurological improvement.

In most of the studies using HIE models, the functional effect of a therapy using NSCs was not evaluated. In only one article, it was shown that NSCs obtained from murine embryonic stem cells improved the spatial memory deficits induced by HI in the P7 mice, when injected 2–3 days after the injury [22]. However, it was not clear if the cells exerted this effect through neural differentiation, replacing of lost neurons, or through a paracrine effect.

Although these studies using models of HIE have demonstrated the migration, proliferation, and differentiation potential of transplanted NSCs, it is still unclear if these cells could result in an improvement in cognitive and motor functions. It needs to be addressed if the transplanted NSCs are able to form proper connections and integrate in the brain for a long time, restoring function.

The Use of Genetically Modified Cells to Deliver Trophic Factors

Since the discovery of nerve growth factor (NGF) by Rita Levi-Montalcini [23], several trophic molecules have been found to have an impact on neuronal survival and growth. The neuroprotective and regenerative capacity of these molecules is still being investigated in many peripheral and CNS disorders. Some of these molecules, such as BDNF, insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF), have already been shown to have neuroprotective and/or regenerative effects in the HI brain [24 –26]. However, the use of cell therapy to deliver these molecules into the brain is a new therapeutic tool that needs to be explored further, since only a few studies have addressed this issue.

Delivery of glial cell line-derived neurotrophic factor (GDNF), for example, by encapsulated baby hamster kidney cells transfected with this factor, grafted into the left hemisphere 2 days before an HI injury, resulted in decreased brain injury and better performance in 3 learning tasks. Serum concentrations of GDNF were higher, even 7 days after the injury, compared to the animals that received non-transfected cells. Moreover, many viable cells were found in the capsules retrieved from animals 7 days after the injury [27,28].

NSCs can also be used to deliver molecules into the brain, as was mentioned above and reviewed by Muller et al. [29].

However, other cell types could be more suitable for gene therapy in the CNS, given their availability and potential to migrate to the brain when injected systemically. For instance, human umbilical cord blood (HUCB) [30], bone marrow, and many other postnatal tissues contain a population of mesenchymal stem cells (MSCs) that can be expanded in vitro [31]. MSCs can mainly differentiate into cells of the mesodermal lineage, such as chondrocytes, osteocytes, and adipocytes. Some groups have also reported the potential of MSCs to differentiate into neurons. However, recently it was reported that rat bone marrow-derived MSCs can express neuronal markers and have a neuronal morphology after the induction of a neuronal transdifferentiation, but do not have other important neuronal properties, such as the firing of action potentials [32]. Bone marrow-derived MSCs home to the ischemic brain in adult rats and this migration depends on the interaction of the chemokine SDF-1α and its receptor CXCR4 [33]. The expression of SDF-1α by astrocytes and endothelial cells increases in the brain of HI mice for at least 7 days after the injury, suggesting that MSCs may also migrate to the HI brain during this time window [34]. Many studies have used genetically modified MSCs to deliver neurotrophic factors to the adult ischemic brain, and in most of them an improvement in functional outcome and a reduction of infarct volume have been demonstrated in these animals [35]. Moreover, it was shown that the growth potential of MSCs after intracranial injection is increased in the neonatal than in the adult brain [36].

In addition, it has been shown that MSCs, while growing in vitro, can secrete many trophic factors into the culture media, even without any genetic manipulation, and it is also possible that the HI brain induces an increase in the production of these factors. In this regard, the cytokine TNF-α increases in the HI brain as early as 3 h after an injury [37], and increases the production of hepatocyte growth factor (HGF), IGF-1, and VEGF by human MSCs [38].

In a recent study, the conditioned medium of adipose MSCs was systemically injected into HI rats, 1 h before or 24 h after the injury. In both cases, the treatment resulted in a decrease in brain damage and in better performance in the Morris water maze tests. Also, it was reported that the neuroprotection could be partially blocked by IGF-1- or BDNF-neutralizing antibodies, showing that both factors contributed to this effect [39].

HUCB, bone marrow, and peripheral blood (PB) contain a population of CD34⁺ cells that can enter the CNS and differentiate into brain macrophages and microglial cells. Lentiviral transfer of genes into these cells does not affect their capacity to populate the brain and differentiate into perivascular or ramified microglia [40]. However, in this study, whole-body irradiation was used to facilitate the grafting of cells in the bone marrow, and this procedure also damages the blood–brain barrier (BBB), increasing the number of cells that manage to infiltrate the brain. On the other hand, the break of the BBB and the release of chemokines during the acute phase of inflammation could also facilitate the infiltration of these cells into the HI brain. Thus, the use of PB, bone marrow, and HUCB cells to deliver genes and consequently their products into the HI brain remains to be elucidated.

In this regard, it was shown that, when HUCB mononuclear cells (HUCBCs) were systemically injected in rodent models of stroke, only a few cells could be found in the brain [41]. However, despite the poor grafting, HUCBC improved behavioral recovery [42], decreased the area of brain infarction [43] and inflammation [44], and improved the regenerative capacity of the brain [45], suggesting a great therapeutic potential for these cells.

Bone Marrow and Umbilical Cord Blood Cells

The use of HUCBCs in animal models of HI encephalopathy was first reported in 2006 [46]. In that study, 1 × 10⁷ cells were injected intraperitoneally 24 h after HI injury in P7 rats. On postnatal day 21 (2 weeks after HI), treated animals had a better motor performance (evaluated by footprint analysis) compared with the untreated ones. Many HUCBCs were found in the ischemic hemisphere of the treated animals. However, the cells did not express neuronal or astrocytic markers. The authors tested only those animals with severe damage (a macroscopic lesion accompanied by cystic changes larger than 4 mm), which was present in 14/20 animals of the treated group and in 11/18 animals of the untreated group, as a way to reduce the normal variability seen in this model.

A recent study found that HUCBCs, even when intravenously injected 7 days after the injury and in a small dose (1.5 × 10⁴ cells), reduced HI-induced deficits in motor asymmetry and motor coordination. The treated animals had increased levels of NGF, GDNF, and BDNF in their brains 3 days after the transplant, indicating a possible paracrine effect of the transplanted cells. When mannitol was administered immediately after the HUCBCs, motor improvement was enhanced and the levels of neurotrophic factors in the brain were increased. Since only a few cells were found in the brains after the treatment, even when mannitol was administered, the authors suggested that this drug facilitates the entry of neurotrophic factors from the circulation into the brain parenchyma, but not of the cells [47].

Recently, our group reported that HUCBCs, when injected 3 h after the injury (2 × 10⁶ cells), improved performance in 2 sensorimotor reflexes that were impaired by HI brain damage. The mechanisms involved in this improvement were a reduction in caspase-3-dependent cell death in the striatum and an anti-inflammatory effect in the cortex. Again, only a few cells could be found in the brain of the treated animals [48].

On the other hand, one group recently reported that intravenous administration of HUCBs has no beneficial effect on the spatial memory deficit induced by HI injury [49].

Taken together, all published studies, with one exception, showed a functional and/or histological improvement of the HI animals after HUCBC transplantation. Moreover, grafting in the brain was not necessary for the functional action of these cells, and even when the cells were found in the brains, there was no evidence of differentiation in neural cells, excluding the possibility of replacement of lost neurons by HUCBCs. Since these cells produce several neurotrophic factors [50] and cytokines [51], it is possible that these factors are released into the circulation, stimulating regenerative events such as angiogenesis and neural plasticity. The neuroprotective and anti-inflammatory effects may also result from the paracrine action of the transplanted cells.

In vitro, HUCBCs protect cortical neurons against glutamate-induced apoptosis by activation of the Akt-signaling pathway [52].

The HUCB mononuclear fraction contains different cell types, and, at present, it is still unknown which cell type(s) are responsible for the therapeutic actions described. HUCB is a rich source of endothelial progenitor cells that can increase neovascularization in mouse models of limb ischemia [53]. In this regard, Taguchi et al. showed that HUCB CD34⁺ cells increase angiogenesis after stroke in the rat [45].

HUCB contains a population of monocytes expressing the cell surface marker CD11b. In vitro, these cells decreased cell viability of microglia under hypoxia, suggesting a possible role of monocytes in the anti-inflammatory effects observed after HUCB transplantation [54]. Moreover, there is a subset of infiltrating monocytes that contributes to recovery after spinal cord injury, through a local anti-inflammatory role [55]. It is tempting to speculate that this population of monocytes is also present in the umbilical cord blood.

Besides the anti-inflammatory effects in the brain [44], HUCBCs may have immunomodulatory effects outside the CNS. It was reported that HUCBC treatment preserved the number of CD8⁺ T cells in the spleen and reduced the stroke-induced reduction in spleen size in rats. HUCBCs altered the expression of cytokines in the spleen of ischemic rats, increasing IL-10 levels and reducing TNF-α production in the treated animals [56].Also, HUCBC treatment reduced systemic inflammation in rodent models of heatstroke and spinal cord injury, decreasing serum levels of TNF-α and increasing IL-10 levels [57,58].

Cord blood MSCs may also have immunomodulatory properties after transplantation [59]. In vitro, MSCs interact with most of the cells of the innate and adaptive immunity, resulting in a complex modulation of the immune response [60].

One possible cell type involved in immunomodulation is the regulatory T cell (Treg). HUCB is enriched in Tregs and it has been shown that HUCB Tregs have a higher suppressor function than adult Tregs [61]. Also, Tregs protect the brain against stroke, reducing the activation of infiltrating and resident inflammatory cells, through the release of the anti-inflammatory cytokine IL-10 [62].

These results, together with the fact that HUCBs promoted an improvement in animal models of stroke and neonatal HI even when transplanted cells were not found in the brain, point to the importance of an immunomodulatory effect in peripheral organs as a mechanism of neuroprotection.

The safety and feasibility of the administration of autologous HUCBCs in term newborns with HIE is being evaluated in a clinical trial being conducted at Duke University (http://www.clinicaltrials.gov/ct2/show/NCT00593242). In this study, children with <6 h of life will be cooled (whole-body hypothermia) and receive the cell therapy, while children with 6 h to 14 d of life will receive only cell therapy. The preliminary efficacy will also be evaluated by neurological examination until 12 months of age and by neuroimaging, compared to historical controls.

In recent years, bone marrow mononuclear cells (BMMCs) and bone marrow-derived MSCs have been shown to have a neuroprotective and regenerative potential in several models of stroke [63]. Importantly, autologous transplantation of these cells in patients after stroke was reported to be safe in phase I clinical trials [64, for review]. Even when injected 1 month after a stroke in rats, bone marrow-derived MSCs improved the functional outcome of the animals [NG_BIB_000_00665]. Moreover, BMMCs injected into the middle cerebral artery of a patient in the chronic phase of stroke (67 days after the onset of symptoms) migrated to the brain parenchyma and were retained in the ischemic hemisphere for at least 2 days [66].

In the model of neonatal HI, the transplantation of bone marrow-derived multipotent adult progenitor cells (MAPCs) 7 days after the injury reduced the loss of hippocampal neurons and improved the motor performance of the animals in the rotarod test. Both the intravenous and the intracerebral delivery of MAPCs resulted in the same effect. However, because the percentage of graft survival was <1% in both cases, the authors could not exclude the possibility of a paracrine effect as the main mechanism involved in the neuroprotection [67]. Also, although most of the injected cells expressed a neuronal marker in the hippocampus of the HI animals, we must be careful before affirming that the cells differentiated into neurons.

An intracardiac injection of human MSCs 72 h after an HI injury resulted in an improvement of sensorimotor deficits measured by the cylinder test. However, the treatment did not affect the brain atrophy. Interestingly, the injected cells could be found throughout the entire brain, in both hemispheres, 6 weeks after transplantation. Most of these cells expressed a microglial marker (OX42) or an astrocytic marker (GFAP) [68].

When injected in the brain, 72 h after an HI injury, mouse MSCs increased the formation of new neurons and OLs in the ischemic hemisphere. The treatment also resulted in a delayed decrease in the area of neuronal loss that may be related to a delayed neuroprotective effect or to cell replacement due to an increase in neurogenesis. Moreover, an anti-inflammatory effect was suggested by a decrease in the number of proliferating microglial cells in the cortex of the treated mice [69].

New studies are needed to confirm that the same effects observed in stroke models could be obtained in the hypoxic–ischemic newborn brain and to determine if bone marrow-derived cells could trigger regenerative processes when transplanted weeks or even months after birth.

In addition, endogenous bone marrow cells can be mobilized to the blood by granulocyte-colony-stimulating factor (G-CSF), resulting in reduced lesion size in transient models of ischemic stroke [70]. This drug is now being tested in phase I/II clinical trials for ischemic stroke. In an animal model of HIE, G-CSF treatment resulted in neuroprotection, inhibiting apoptotic cell death [71]. Moreover, G-CSF may also promote an increase in neurogenesis and have direct neuroprotective effects.

Endogenous NSC Response to Injury

During brain development, a secondary proliferative zone is formed just below the ventricular zone: the SVZ. This region persists after birth and generates new neurons and glial cells throughout life.

Several studies have reported an increase in SVZ proliferation and neurogenesis after neonatal HI in the rat [72 –75].

It was reported that the hypoxic–ischemic SVZ generated twice as many neurospheres as the control SVZ. Also, the percentage of multipotential neurospheres and the number of symmetrical cell divisions were increased after HI, indicating that the injury affected intrinsic properties of the neural stem/precursor cells [76]. One possible candidate to modulate the expansion of the NSC pool after HI is the leukemia inhibitory factor (LIF), up-regulated in the SVZ in the first 2 days after the injury. In vitro, LIF increases the number and size of SVZ-derived neurospheres [77].

In the hypoxic–ischemic neocortex, neuronal death occurs in a columnar pattern, and these columns are populated by reactive astrocytes and microglia in the first days after the injury. Recently, it was reported that newborn neurons migrated from the SVZ to the damaged neocortex, occupying these cell-sparse columns, and differentiated into neocortical neurons expressing calretinin. Surprisingly, the increased production of new neurons persisted for at least 5 months after the injury [78]. However, the authors also showed that around 85% of the newborn neurons died before maturation. This limitation is a potential target for future therapies aiming to increase the survival and integration of endogenous newly formed neurons in the injured cortex.

In the rat striatum, >95% of the neurons are GABAergic medium-sized spiny projecting neurons, and <5% are interneurons that can be divided into 4 populations, based on the expression of calretinin, parvalbumin, somatostatin, or choline acetyltransferase. However, only calretinin-expressing interneurons were formed in the hypoxic–ischemic striatum, constituting an important limitation for the regeneration of this brain region [79].

Modulation of the endogenous capacity to form new neurons is a new challenge, and promising treatments may be developed to overcome the limitations of this self-repair mechanism. In this regard, it was shown that intracerebroventricular administration of bFGF factor increased the number of newly born neurons in the SVZ after bilateral occlusion of the common carotid arteries of postnatal day 3 rats [80]. However, the functional effect of this treatment was not evaluated.

In the mouse hippocampus, basal proliferation and neurogenesis are higher in the immature (postnatal day 9) than in juvenile (postnatal day 21) animals. However, after an HI injury, neurogenesis in the dentate gyrus was increased only in the juvenile mice, reaching the same level of the non-injured immature hippocampus. The juvenile hippocampus also had increased microglial proliferation and higher levels of the chemokine MCP-1 and the cytokine IL-18 [81]. It would be interesting to know if the increased inflammation in the juvenile brain triggered the regenerative response in the hippocampus. On the other hand, one study found increased hippocampal neurogenesis after HI in the postnatal day 7 mouse [82].

Up to now, hypothermia is the most successful treatment to reduce long-term effects of moderate and severe HIE in preclinical and clinical studies. However, one study showed that hypothermia decreased the number of proliferating cells in the subgranular zone of the hippocampus, but not in the SVZ of postnatal day 7 rats [83]. This study is preliminary, and did not investigate if the same effect occurs when HI animals are cooled or if it is transient, but the study does raise at least 2 questions: (1) How would a possible treatment for HIE interfere with the regenerative capacity of the newborn brain? and (2) How would hypothermia or any other possible drug treatment for HIE interfere with a cell therapy?

Erythropoietin is a good example for the first question. This drug had a neuroprotective effect in several preclinical studies, and improved the neurological outcome of children with moderate HIE in a recent clinical trial [84]. Erythropoietin has also been shown to increase the formation of new neurons after an HI injury in rats [85].

Encephalopathy of Prematurity

The term encephalopathy of prematurity was proposed recently to describe the pattern of brain injury that commonly occurs in premature infants, especially in those with a very low birth weight (VLBW; ≤1,500 g), a group that represents 1.5% of all live births. Periventricular leukomalacia (PVL), a distinct form of cerebral white matter injury, is the classical form of brain injury in these children, and in many cases is accompanied by neuronal/axonal damage in the white matter, cerebral cortex, brainstem, basal ganglia, and/or cerebellum [86].

However, despite the increasing importance of PVL, given that the number of surviving VLBW children increases each year, studies evaluating the potential of stem cell therapies for remyelination in models of PVL are lacking.

It was shown that HUCBCs protect OLs, in vitro, in a model of oxygen glucose deprivation [87]. However, in that study, 60% of the OLs had a mature phenotype, and it is still unknown whether HUCBCs could also rescue immature OLs, especially late OL progenitors, which are present in abundance in the periventricular white matter during the developmental window of vulnerability for PVL [88].

Human oligodendrocyte progenitor cells (OPCs) of both adult and fetal origin have been transplanted in a mouse model of congenital demyelination (the shiverer mouse, deficient in myelin basic protein). These cells migrated throughout the brain and differentiated into astrocytes in the gray matter and into OLs in the white matter. The latter were able to remyelinate the shiverer brain, with adult OPCs ensheathing more axons per donor cell than the fetal OPCs [89].

In a model of HI white matter injury in the P3 rat, it was found that the degeneration of preoligodendrocytes (preOLs) occurred in 2 phases: acute caspase-3-independent cell death and delayed caspase-3-dependent cell death. Later, there was proliferation and accumulation of preOLs that displayed an arrest of maturation and failed to initiate myelination [90]. If this arrest results from an alteration in the microenvironment (eg, the presence of hyaluronic acid in the glial scar), it is possible that OPCs transplanted into the HI brain could also fail to differentiate into mature OLs. In this case, new strategies should be tested to promote the proper differentiation of the endogenous preOLs that accumulate after the injury and to prevent the possible arrest of maturation of transplanted OPCs.

However, transplantation of OPCs alone may not be enough to treat or avoid all the cognitive, motor, and behavioral deficits in the encephalopathy of prematurity, since neuronal injury has also recently been found to be present in these children.

Conclusions

At present, many different cell types have been tested in rodent models of neonatal HIE. However, several questions remain to be investigated in order to envision the translation of these results to clinical trials. For instance, it is important to determine the best cell type (s), the route of administration, the number of cells, and the timing for administration of the cells. Also, the mechanisms involved in the therapeutic effects should be carefully investigated in order to provide new insights into how the therapy can be improved by genetic modifications.