Trophic factors and cell therapy to stimulate brain repair after ischaemic stroke

Brain repair involves a compendium of natural mechanisms that are activated following stroke. From a therapeutic viewpoint, reparative therapies that encourage cerebral plasticity are needed. In the last years, it has been demonstrated that modulatory treatments for brain repair such as trophic factor- and stem cell-based therapies can promote neurogenesis, gliogenesis, oligodendrogenesis, synaptogenesis and angiogenesis, all of which having a beneficial impact on infarct volume, cell death and, finally, and most importantly, on the functional recovery. However, even when promising results have been obtained in a wide range of experimental animal models and conditions these preliminary results have not yet demonstrated their clinical efficacy. Here, we focus on brain repair modulatory treatments for ischaemic stroke, that use trophic factors, drugs with trophic effects and stem cell therapy. Important and still unanswered questions for translational research ranging from experimental animal models to recent and ongoing clinical trials are reviewed here.     

Increased NADPH oxidase-derived superoxide is involved in the neuronal cell death induced by hypoxia-ischemia in neonatal hippocampal slice cultures

Neonatal brain hypoxia-ischemia (HI) results in neuronal cell death. Previous studies indicate that reactive oxygen species, such as superoxide, play a key role in this process. However, the cellular sources have not been established. In this study we examine the role of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in neonatal HI brain injury and elucidate its mechanism of activation. Rat hippocampal slices were exposed to oxygen glucose deprivation (OGD) to mimic the conditions seen in HI. Initial studies confirmed an important role for NADPH oxidase-derived superoxide in the oxidative stress associated with OGD. Further, the OGD-mediated increase in apoptotic cell death was inhibited by the NADPH oxidase inhibitor apocynin. The activation of NADPH oxidase was found to be dependent on the p38 mitogen-activated protein kinase-mediated phosphorylation and activation of the p47(phox) subunit. Using an adeno-associated virus antisense construct to selectively decrease p47(phox) expression in neurons showed that this led to inhibition of both the increase in superoxide and the neuronal cell death associated with OGD. We also found that NADPH oxidase inhibition in a neonatal rat model of HI or scavenging hydrogen peroxide reduced brain injury. Thus, we conclude that activation of the NADPH oxidase complex contributes to the oxidative stress during HI and that therapies targeted against this complex could provide neuroprotection against the brain injury associated with neonatal HI.     

Limited role of the c-Jun N-terminal kinase pathway in a neonatal rat model of cerebral hypoxia–ischemia

D-JNKI1, a cell-permeable peptide inhibitor of the c-Jun N-terminal kinase (JNK) pathway, has been shown to be a powerful neuroprotective agent after focal cerebral ischemia in adult mice and young rats. We have investigated the potential neuroprotective effect of D-JNKI1 and the involvement of the JNK pathway in a neonatal rat model of cerebral hypoxia–ischemia (HI). Seven-day-old rats underwent a permanent ligation of the right common carotid artery followed by 2 h of hypoxia (8% oxygen). Treatment with D-JNKI1 (0.3 mg/kg intraperitoneally) significantly reduced early calpain activation, late caspase 3 activation and, in the thalamus, autophagosome formation, indicating an involvement of JNK in different types of cell death: necrotic, apoptotic, and autophagic. However, the size of the lesion was unchanged. Further analysis showed that neonatal HI induced an immediate decrease in JNK phosphorylation (reflecting mainly JNK1 phosphorylation) followed by a slow progressive increase (including JNK3 phosphorylation 54 kDa), whereas c-jun and c-fos expression were both strongly activated immediately after HI. In conclusion, unlike in adult ischemic models, JNK is only moderately activated after severe cerebral HI in neonatal rats and the observed positive effects of D-JNKI1 are insufficient to give neuroprotection. Thus, for perinatal asphyxia, D-JNKI1 can only be considered in association with other therapies.     

Mannitol facilitates neurotrophic factor up‐regulation and behavioural recovery in neonatal hypoxic‐ischaemic rats with human umbilical cord blood grafts

We recently demonstrated that blood–brain barrier permeabilization using mannitol enhances the therapeutic efficacy of systemically administered human umbilical cord blood (HUCB) by facilitating the entry of neurotrophic factors from the periphery into the adult stroke brain. Here, we examined whether the same blood–brain barrier manipulation approach increases the therapeutic effects of intravenously delivered HUCB in a neonatal hypoxic‐ischaemic (HI) injury model. Seven‐day‐old Sprague–Dawley rats were subjected to unilateral HI injury and then at day 7 after the insult, animals intravenously received vehicle alone, mannitol alone, HUCB cells (15k mononuclear fraction) alone or a combination of mannitol and HUCB cells. Behavioural tests at post‐transplantation days 7 and 14 showed that HI animals that received HUCB cells alone or when combined with mannitol were significantly less impaired in motor asymmetry and motor coordination compared with those that received vehicle alone or mannitol alone. Brain tissues from a separate animal cohort from the four treatment conditions were processed for enzyme‐linked immunosorbent assay at day 3 post‐transplantation, and revealed elevated levels of GDNF, NGF and BDNF in those that received HUCB cells alone or when combined with mannitol compared with those that received vehicle or mannitol alone, with the combined HUCB cells and mannitol exhibiting the most robust neurotropic factor up‐regulation. Histological assays revealed only sporadic detection of HUCB cells, suggesting that the trophic factor–mediated mechanism, rather than cell replacement per se, principally contributed to the behavioural improvement. These findings extend the utility of blood–brain barrier permeabilization in facilitating cell therapy for treating neonatal HI injury.     

血糖水平对缺氧缺血新生大鼠体重和脑重的影响观察

目的　观察不同血糖水平及缺氧缺血 (hypoxicischemia ,HI)前后血糖对缺氧缺血新生大鼠体重和脑重的影响。方法　通过制备缺氧缺血新生大鼠合并高低血糖模型 ,分别在脑HI后 2、2 4、48、72h和 7d共 5个时段 ,分别在断头前测体重和断头后取脑称脑重 ,并辅以免疫组化分析HI后 2 4h时段各组脑内葡萄糖转运蛋白 1(GLUT1)及葡萄糖转运蛋白 3 (GLUT3 )合成量的变化。结果　 2 4h时段HI组、HI前低血糖组、HI后低血糖组体重呈显著意义 ,低于正常组 ;2 4h时段缺氧缺血前后低血糖脑重均呈显著意义 ,低于其他各组 ,7d时段I前重高血糖组脑重高于HI组及HI前低血糖组。HI前重高血糖组HI后 2 4h在皮质部位GLUT1的合成量显著高于其他各组。HI前低血糖组HI后 2 4h在皮质部位GLUT3的合成量显著低于其他各组。结论　HI前低血糖对新生大鼠体重、脑重增长不利 ,HI前重高血糖有可能缓解HI引起的体重、脑重的减轻而显示一定的保护作用     

Hypoxia Ischemia-Mediated Cell Death in Neonatal Rat Brain

The examination of Bcl-2-associated X protein (Bax) protein’s role in the activation of cognate nuclear, mitochondrial and ER cell death signaling cascades and the resulting effects on cell death phenotype in the brain after neonatal hypoxia-ischemia (HI) requires an understanding of neonatal HI insult and progression, as well as, its dysfunctional outcomes. In addition, knowledge of key concepts of oxidative stress, a major injurious component of HI, and the different cell death phenotypes (i.e. apoptosis and necrosis) will aid the design of appropriate useful experimental paradigms. Here we discuss organelle cell death signaling cascades in the context of the different cell death phenotypes associated with animal models of neonatal hypoxia ischemia and tissue culture models used in the study of hypoxia ischemia, focusing on the intracellular shifts of the Bcl-2 associated X protein (Bax) in the hypoxic brain. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Glucose and Intermediary Metabolism and Astrocyte–Neuron Interactions Following Neonatal Hypoxia–Ischemia in Rat

Neonatal hypoxia–ischemia (HI) and the delayed injury cascade that follows involve excitotoxicity, oxidative stress and mitochondrial failure. The susceptibility to excitotoxicity of the neonatal brain may be related to the capacity of astrocytes for glutamate uptake. Furthermore, the neonatal brain is vulnerable to oxidative stress, and the pentose phosphate pathway (PPP) may be of particular importance for limiting this kind of injury. Also, in the neonatal brain, neurons depend upon de novo synthesis of neurotransmitters via pyruvate carboxylase in astrocytes to increase neurotransmitter pools during normal brain development. Several recent publications describing intermediary brain metabolism following neonatal HI have yielded interesting results: (1) Following HI there is a prolonged depression of mitochondrial metabolism in agreement with emerging evidence of mitochondria as vulnerable targets in the delayed injury cascade. (2) Astrocytes, like neurons, are metabolically impaired following HI, and the degree of astrocytic malfunction may be an indicator of the outcome following hypoxic and hypoxic-ischemic brain injury. (3) Glutamate transfer from neurons to astrocytes is not increased following neonatal HI, which may imply that astrocytes fail to upregulate glutamate uptake in response to the massive glutamate release during HI, thus contributing to excitotoxicity. (4) In the neonatal brain, the activity of the PPP is reduced following HI, which may add to the susceptibility of the neonatal brain to oxidative stress. The present review aims to discuss the metabolic temporal alterations observed in the neonatal brain following HI.     

Evaluation of acetylcholinesterase and adenosine deaminase activities in brain and erythrocytes and proinflammatory cytokine levels in rats submitted to neonatal hypoxia-ischemia model

Perinatal hypoxic-ischemic (HI) brain injury is a common problem with severe neurologic sequelae. The definitive brain injury is a consequence of pathophysiological mechanisms that begin at the moment of HI insult and may extend for days or weeks. In this context, the inflammatory response and the formation of reactive oxygen species seem to play a key role during evolution of brain damage after injury. Thus, the aim of this study was to describe the chronological sequence of acetylcholinesterase (AChE) activity and the lipid peroxidation changes in the cerebral cortex using the classic model of neonatal HI. Furthermore, the erythrocyte AChE and adenosine deaminase (ADA) activities as well as the serum levels of proinflammatory cytokines were assessed. We observed that neonatal HI caused an increase of lipid peroxidation immediately after HI insult, which remained for several days afterward. There was a time-related change in the AChE activity in the cerebral cortex and the same was observed in erythrocyte AChE and ADA activities. In addition, immediately after HI, ADA activity showed a strong positive correlation with all proinflammatory cytokines assessed. Together, these findings may help the understanding of some mechanism related to the pathophysiology of neonatal HI, therefore highlighting the putative therapeutic targets to minimize brain injury and enhance recovery.     

Multi-Modal Assessment of Long-Term Erythropoietin Treatment after Neonatal Hypoxic-Ischemic Injury in Rat Brain

Erythropoietin (EPO) has been recognized as a neuroprotective agent. In animal models of neonatal brain injury, exogenous EPO has been shown to reduce lesion size, improve structure and function. Experimental studies have focused on short course treatment after injury. Timing, dose and length of treatment in preterm brain damage remain to be defined. We have evaluated the effects of high dose and long-term EPO treatment in hypoxic-ischemic (HI) injury in 3 days old (P3) rat pups using histopathology, magnetic resonance imaging (MRI) and spectroscopy (MRS) as well as functional assessment with somatosensory-evoked potentials (SEP). After HI, rat pups were assessed by MRI for initial damage and were randomized to receive EPO or vehicle. At the end of treatment period (P25) the size of resulting cortical damage and white matter (WM) microstructure integrity were assessed by MRI and cortical metabolism by MRS. Whisker elicited SEP were recorded to evaluate somatosensory function. Brains were collected for neuropathological assessment. The EPO treated animals did not show significant decrease of the HI induced cortical loss at P25. WM microstructure measured by diffusion tensor imaging was improved and SEP response in the injured cortex was recovered in the EPO treated animals compared to vehicle treated animals. In addition, the metabolic profile was less altered in the EPO group. Long-term treatment with high dose EPO after HI injury in the very immature rat brain induced recovery of WM microstructure and connectivity as well as somatosensory cortical function despite no effects on volume of cortical damage. This indicates that long-term high-dose EPO induces recovery of structural and functional connectivity despite persisting gross anatomical cortical alteration resulting from HI.     

Plasticity of Neurons and Glia Following Neonatal Hypoxic-Ischemic Brain Injury in Rats

Periventricular white matter injury in premature infants is linked to chronic neurological dysfunction. Periventricular white matter injury is caused by many mechanisms including hypoxia-ischemia (HI). Animal models of HI in the neonatal rodent brain can replicate some important features of periventricular white matter injury. Most rodent studies have focused upon early cellular and tissue events following unilateral neonatal HI that is elicited by unilateral carotid artery ligation and followed by timed exposure to moderate hypoxia. Milder hypoxic-ischemic insults elicit preferential white matter injury. Little information is available about long-term cellular effects of unilateral HI. One month after unilateral neonatal hypoxia ischemia, we show that all the components for structural reorganization of the brain are present in moderately injured rats. These components in the injured side include extensive influx of neurites, axonal and dendritic growth cones, abundant immature synapses, and myelination of many small axons. Surprisingly, this neural recovery is often found in and adjacent to cysts that have the ultrastructural features of bone extracellular matrix. In contrast, brains with severe hypoxia ischemia one month after injury still undergo massive neuronal degeneration. While massive destruction of neurons and glia are striking events shortly after brain HI, neural cells re-express their intrinsic properties and attempt an anatomical recovery long after injury. Special issue dedicated to Anthony Campagnoni.     

CREB activation in the rapid, intermediate, and delayed ischemic preconditioning against hypoxic–ischemia in neonatal rat

Ischemic preconditioning (IP) is a defense program in which exposure to sublethal ischemia followed by a period of reperfusion results in subsequent resistance to severe ischemic insults. Very few in vivo IP models have been established for neonatal brain. We examined whether rapid, intermediate, and delayed IP against hypoxic–ischemia (HI) could be induced in neonatal brain, and if so, whether the IP involved phosphorylation of cAMP response element-binding protein (pCREB) after HI. Postnatal day 7 rat pups were subjected to HI at 2 h (2-h IP), 6 h (6-h IP), or 22 h (22-h IP) after IP. We found all three IP groups had significantly reduced neuronal damage and TUNEL-(+) cells 24 h post-HI than no-IP group. Compared with control, the no-IP group had significant decreases of pCREB and mitochondria Bcl-2 levels in the ipsilateral cortex 24 h post-HI. In contrast, the three IP groups had increased pCREB and mitochondria Bcl-2 levels, and significant differences were found between three IP and no-IP groups. The increases of cleavage of caspase-3 and poly (ADP-ribose) polymerase and of cells with nuclear apoptosis inducing factor post-HI in no-IP group were all significantly reduced in three IP groups. The increases of caspase-3 and calpain-mediated proteolysis of α-spectrin post-HI were significantly reduced only in 22-h IP group. Furthermore, all three IP groups had long-term neuroprotection at behavioral and pathological levels compared with no-IP group. In conclusion, IP, rapid, intermediate, or delayed, in neonatal rat brain activates CREB, up-regulates Bcl-2, induces extensive brakes on caspase-dependent and -independent apoptosis after HI, and provides long-term neuroprotection.     

Intranasal Mesenchymal Stem Cell Treatment for Neonatal Brain Damage: Long-Term Cognitive and Sensorimotor Improvement

Mesenchymal stem cell (MSC) administration via the intranasal route could become an effective therapy to treat neonatal hypoxic-ischemic (HI) brain damage. We analyzed long-term effects of intranasal MSC treatment on lesion size, sensorimotor and cognitive behavior, and determined the therapeutic window and dose response relationships. Furthermore, the appearance of MSCs at the lesion site in relation to the therapeutic window was examined. Nine-day-old mice were subjected to unilateral carotid artery occlusion and hypoxia. MSCs were administered intranasally at 3, 10 or 17 days after hypoxia-ischemia (HI). Motor, cognitive and histological outcome was investigated. PKH-26 labeled cells were used to localize MSCs in the brain. We identified 0.5×10⁶ MSCs as the minimal effective dose with a therapeutic window of at least 10 days but less than 17 days post-HI. A single dose was sufficient for a marked beneficial effect. MSCs reach the lesion site within 24 h when given 3 or 10 days after injury. However, no MSCs were detected in the lesion when administered 17 days following HI. We also show for the first time that intranasal MSC treatment after HI improves cognitive function. Improvement of sensorimotor function and histological outcome was maintained until at least 9 weeks post-HI. The capacity of MSCs to reach the lesion site within 24 h after intranasal administration at 10 days but not at 17 days post-HI indicates a therapeutic window of at least 10 days. Our data strongly indicate that intranasal MSC treatment may become a promising non-invasive therapeutic tool to effectively reduce neonatal encephalopathy.     

Remote Neurodegeneration: Multiple Actors for One Play

Remote neurodegeneration significantly influences the clinical outcome in many central nervous system (CNS) pathologies, such as stroke, multiple sclerosis, and traumatic brain and spinal cord injuries. Because these processes develop days or months after injury, they are accompanied by a therapeutic window of opportunity. The complexity and clinical significance of remote damage is prompting many groups to examine the factors of remote degeneration. This research is providing insights into key unanswered questions, opening new avenues for innovative neuroprotective therapies. In this review, we evaluate data from various remote degeneration models to describe the complexity of the systems that are involved and the importance of their interactions in reducing damage and promoting recovery after brain lesions. Specifically, we recapitulate the current data on remote neuronal degeneration, focusing on molecular and cellular events, as studied in stroke and brain and spinal cord injury models. Remote damage is a multifactorial phenomenon in which many components become active in specific time frames. Days, weeks, or months after injury onset, the interplay between key effectors differentially affects neuronal survival and functional outcomes. In particular, we discuss apoptosis, inflammation, oxidative damage, and autophagy—all of which mediate remote degeneration at specific times. We also review current findings on the pharmacological manipulation of remote degeneration mechanisms in reducing damage and sustaining outcomes. These novel treatments differ from those that have been proposed to limit primary lesion site damage, representing new perspectives on neuroprotection.     

Overexpression of HIF-1α in mesenchymal stem cells contributes to repairing hypoxic-ischemic brain damage in rats

Preclinical researches on mesenchymal stem cells (MSCs) transplantation, which is used to treat hypoxic-ischemic (HI) brain damage, have received inspiring achievements. However, the insufficient migration of active cells to damaged tissues has limited their potential therapeutic effects. There are some evidences that hypoxia inducible factor-1 alpha (HIF-1α) promotes the viability and migration of the cells. Here, we aim to investigate whether overexpression of HIF-1α in MSCs could improve the viability and migration capacity of cells, and its therapeutic efficiency on HI brain damage. In the study, MSCs with HIF-1α overexpression was achieved by recombinant lentiviral vector and transplanted to the rats subsequent to HI. Our data indicated that overexpression of HIF-1α promoted the viability and migration of MSCs, HIF-1α overexpressed MSCs also had a stronger therapeutic efficiency on HI brain damaged treatment by mitigating the injury on behavioral and histological changes evoked by HI insults, accompanied with more MSCs migrating to cerebral damaged area. This study demonstrated that HIF-1α overexpression could increase the MSCs’ therapeutic efficiency in HI and the promotion of the cells’ directional migration to cerebral HI area by overexpression may be responsible for it, which showed that transplantation of MSCs with HIF-1α overexpression is an attractive therapeutic option to treat HI-induced brain injury in the future.     

Adult stem cells and bioengineering strategies for the treatment of cerebral ischemic stroke

The adult central nervous system (CNS) contains a population of neural stem cells, yet unlike many other tissues, has a very limited capacity for self-repair. Promoting tissue repair and functional recovery following CNS injury or disease is a high priority as there are currently no effective treatments towards this end for the treatment of disorders such as stroke, traumatic brain injury and spinal cord injury. Recent advances in stem cell biology have offered a number of enticing potential avenues and we will discuss these possibilities along with the associated challenges as they pertain to stroke. We will consider exogenous therapies involving the transplantation of adult stem cells, and the mobilization of endogenous stem cells, as well as drug delivery and tissue engineering strategies that enhance and complement the cell based strategies.     

MMP-9 Inhibition: a Therapeutic Strategy in Ischemic Stroke

Ischemic stroke is a leading cause of disability worldwide. In cerebral ischemia there is an enhanced expression of matrix metallo-proteinase-9 (MMP-9), which has been associated with various complications including excitotoxicity, neuronal damage, apoptosis, blood–brain barrier (BBB) opening leading to cerebral edema, and hemorrhagic transformation. Moreover, the tissue plasminogen activator (tPA), which is the only US-FDA approved treatment of ischemic stroke, has a brief 3 to 4 h time window and it has been proposed that detrimental effects of tPA beyond the 3 h since the onset of stroke are derived from its ability to activate MMP-9 that in turn contributes to the breakdown of BBB. Therefore, the available literature suggests that MMP-9 inhibition can be of therapeutic importance in ischemic stroke. Hence, combination therapies of MMP-9 inhibitor along with tPA can be beneficial in ischemic stroke. In this review we will discuss the current status of various strategies which have shown neuroprotection and extension of thrombolytic window by directly or indirectly inhibiting MMP-9 activity. In the introductory part of the review, we briefly provide an overview on ischemic stroke, commonly used models of ischemic stroke and a role of MMP-9 in ischemia. In next part, the literature is organized as various approaches which have proven neuroprotective effects through direct or indirect decrease in MMP-9 activity, namely, using biotherapeutics, involving MMP-9 gene inhibition using viral vectors; using endogenous inhibitor of MMP-9, repurposing of old drugs such as minocycline, new chemical entities like DP-b99, and finally other approaches like therapeutic hypothermia.     

The immunology of stroke: from mechanisms to translation

Immunity and inflammation are key elements of the pathobiology of stroke, a devastating illness second only to cardiac ischemia as a cause of death worldwide. The immune system participates in the brain damage produced by ischemia, and the damaged brain, in turn, exerts an immunosuppressive effect that promotes fatal infections that threaten the survival of people after stroke. Inflammatory signaling is involved in all stages of the ischemic cascade, from the early damaging events triggered by arterial occlusion to the late regenerative processes underlying post-ischemic tissue repair. Recent developments have revealed that stroke engages both innate and adaptive immunity. But adaptive immunity triggered by newly exposed brain antigens does not have an impact on the acute phase of the damage. Nevertheless, modulation of adaptive immunity exerts a remarkable protective effect on the ischemic brain and offers the prospect of new stroke therapies. As immunomodulation is not devoid of deleterious side effects, a better understanding of the reciprocal interaction between the immune system and the ischemic brain is essential to harness the full therapeutic potential of the immunology of stroke.     

Neurotrophin-3, TNF-alpha and IL-6 relations in serum and cerebrospinal fluid of ischemic stroke patients

Pro-inflammatory cytokines and neurotrophins in the central nervous system (CNS) have been recognized as mediators of both neurodegenerative and neuroprotective mechanisms in a number of CNS pathologies. A rapid, sustained elevation of these molecules was recently reported after traumatic and ischemic brain injury. Inflammatory mechanisms and immune activation have been hypothesized to play a role in the pathogenesis of cerebral ischemia. Stroke is the third largest cause of death next to heart disease and cancer in the world, and it is an important cause of death and disability in developed countries. Role of excitatory amino acids receptors activation, calcium overload, nitric oxide and oxidative stress in the pathogenesis of ischemic brain damage is well established. Stroke may modulate peripheral neurotrophic factors levels. In experimental animal models, neurotrophin-3 (NT-3) has been shown to be produced by glial cells as an adaptability response to hypoxia. In spite of substantial research and significant number of neuroprotective drugs that have been developed to limit ischemic brain damage and to improve the outcome for stroke patients, no specific therapy for stroke is available. The neurotrophins have been proposed as therapeutic agents for the treatment of neurodegenerative disorders and ischemic injury. In the present work, we investigated the possible correlation of NT-3 with tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the serum and cerebrospinal fluid (CSF) from patients with ischemic stroke (IS).