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• Neurotrophic factors probably regulate the extent of neuronal death, axonal sprouting, and synaptic rearrangements that occur at the site of ischemic injury.
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• The major role of neurotrophic factors is in the promotion of neural regeneration following ischemic injury.
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• Neuroprotective role of brain-derived neurotrophic factor in focal cerebral ischemia includes protective effects against glutamate toxicity and attenuation of apoptosis.
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• Changes in neurotrophic factors are among the early biomarkers of ischemic stroke and are the basis of neuroprotective therapeutic approaches in cerebral ischemia.
Ischemic stroke as a central nervous system injury. There is considerable evidence that neurotrophic factors are involved in the response to injury to the nervous system, whether it is caused by mechanical trauma, ischemia, hypoxia, or metabolic insults such as hypoglycemic coma resulting in neuronal loss. Whereas increased nerve growth factor and brain-derived neurotrophic factor with decreased neurotrophin-3 mRNA are observed following afferent stimulation, a different pattern of neurotrophin mRNA expression occurs following certain injuries wherein neurotrophin-3 mRNA is increased, and brain-derived neurotrophic factor mRNA is relatively decreased. All these changes are mediated by glutamate receptor activation and increased intracellular calcium.
Role of neurotrophic factors in central nervous system injury. In addition to neurotrophins, glial-associated factors, including basic fibroblast growth factor, insulin-like growth factor-1, transforming growth factor-beta, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 are more highly expressed at the site of injury. These factors probably regulate the extent of neuronal death, axonal sprouting, and synaptic rearrangements that occur in these regions. Many of the glial-associated factors, in contrast to the neuronal-derived neurotrophic factors, also have mitogenic effects on glial cells. Data from mouse models of stroke indicate that glial cell line-derived neurotrophic factor potently induces long-term neurologic recovery, peri-infarct brain remodeling, and contralesional neuroplasticity, which are associated with the fine-tuned regulation of axonal growth inhibitors and guidance molecules (02).
Molecular mechanisms in ischemic stroke. Several mechanisms are involved in the pathogenesis of neurologic deficits due to ischemic cerebrovascular disease. An increasing amount of experimental evidence indicates that the 2 broad mechanisms (excessive activation of glutamate receptors and oxidative stress) converge and provide a final common pathway for cell vulnerability in the brain during ischemia. Molecular mechanisms relevant to neurotrophic factors are
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• Altered gene regulation or expression in cerebral ischemia
• Increase of transcription of mRNA for heat shock proteins
• Proto-oncogene transcription factor c-fos
• Promotion of angiogenesis
• Stimulation of local synthesis of neurotrophic factors
• Stimulation of synthesis of cytokines: interleukin-1 beta and tumor necrosis factor-alpha
There is expression of a range of genes in cerebral ischemia that may have a beneficial or detrimental effect on the evolution of neuronal injury. The first set of genes to be activated following neuronal injury in focal cerebral ischemia is the c-fos and c-jun complex that are involved in the induction of target genes that regulate cell growth and differentiation. According to the excitotoxic hypothesis, ischemic neuronal death is induced by the release of excitatory amino acid glutamate. Activation of the N-methyl-D-aspartate receptor-operated and voltage-sensitive calcium channels cause calcium influx that activates degrading enzymes leading to disintegration of nuclear and cell membranes and generation of oxygen free radicals. Calcium influx also induces expression of c-fos. Damage, repair, and transcription of the c-fos gene occur during cerebral ischemia. Fos peptide, one of the components of activator protein 1, activates nerve growth factor and repair mechanisms.
Changes in neurotrophic factor associated with cerebral ischemia. These are shown in Table 1. Changes in neurotrophic factors are among the early biomarkers of ischemic stroke. Some of these are the basis of neuroprotective therapeutic approaches in cerebral ischemia.
Table 1. Changes in Neurotrophic Factors in Experimental Stroke or Autopsy Brain Examination of Stroke Patients
Brain-derived neurotrophic factor
Expression is increased but varies in different parts of the brain.
These observations are consistent with the neuroprotective role of brain-derived neurotrophic factor in cortical neurons.
Basic fibroblast growth factor
The level of serum basic fibroblast growth factor increases significantly in the patients with acute cerebral infarction and correlate with clinical improvement.
Helpful in estimating the size of infarct at acute stage of cerebral infarction.
Ciliary neurotrophic factor
Increase in the ischemic hemisphere.
This supports the survival and regeneration of neurons in the damaged area.
Insulin-like growth factor-1
Increase within ischemic neurons indicating that it participates in reaction to ischemic injury.
Expression of neuregulin-1 in neurons is induced in the ischemic penumbra by focal stroke in the rat.
Neuregulin-1 protects neurons from delayed, ischemia-induced cell death by inhibiting pro-inflammatory responses.
The 3 major hypotheses for the functional effects of cerebral insult-induced neurotrophin changes are: (1) protection against neuronal damage, (2) stimulation of neuronal sprouting, and (3) synaptic reorganization.
Potential stroke therapies have 3 major aims:
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(1) Restoration of the patency of the obstructed arteries
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(2) Neuroprotection to limit the brain damage caused by lack of blood circulation or reperfusion
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(3) Promotion of neuronal regeneration and recovery following ischemic insult
Neurotrophic factors are not relevant to the first aim, but they do have some neuroprotective function. Their major role is in the promotion of neural regeneration. The role of various neurotrophic factors varies following ischemic stroke. For example, insulin-like growth factor-1 can induce proliferation of neural stem cells in the presence of fibroblast growth factor-2 (FGF-2) and promote differentiation in its absence. It is important to understand the role of each neurotrophic factor in optimizing the endogenous repair following stroke.
Potential role of neurotrophic factors in the treatment of cerebral ischemia. The viability of neuronal cells in the "penumbra" zone has provided the possibility to rescue these cells after focal ischemia. At least 4 growth factor superfamilies have been shown to reduce cell death when introduced directly into the brains of animal models of cerebral ischemia: (1) neurotrophins, (2) fibroblast growth factors, (3) transforming growth factor-beta 1, and (4) insulin-like growth factors. The hypoxic-ischemic encephalopathy seen in survivors of perinatal asphyxia is a frequently encountered clinical problem. Secretion of neurotrophic factors—glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor—from choroid transplants improves behavioral performance and decreases the volume of infarction in rodent stroke models. Delivery of neurotrophic factors to the brain by gene therapy for the treatment of stroke is described in a separate article on this topic.
Fibroblast growth factor. Basic fibroblast growth factor is a potent dilator of systemic blood vessels and cerebral pial arterioles. Intravenous basic fibroblast growth factor can cross the damaged blood-brain barrier and exert a direct trophic effect on the ischemic brain tissue. Intracisternal basic fibroblast growth factor has also been shown to enhance behavioral recovery following focal cerebral infarction in the rat. The mechanism of infarct reduction may include direct cytoprotective and vasoactive effects. No angiogenesis has been demonstrated in any of the studies, but basic fibroblast growth factor is known to have angiogenic properties. Although the exact mechanisms of the beneficial effect of basic fibroblast growth factor in ischemic stroke have not been proven, even delayed administration enhances functional recovery. The mechanism of action of basic fibroblast growth factor as a neuroprotective agent involves activation of survival signals that, in turn, enhance antiapoptotic proteins, antioxidant enzymes, and calcium-binding proteins. There are differences in the response of aged versus young adult rats to fibroblast growth factor in cerebral ischemia; unlike in young adult ischemic rats, only few newly generated cells migrate into the infarcted region in aged brain.
Basic fibroblast growth factor has a synergistic effect with citicoline (cytidine 5'-diphosphate choline) as a neuroprotective in animal models of focal cerebral ischemia. Citicoline is used as a treatment for stroke in some countries, but phase 3 trials have not yet proven its efficacy.
Brain-derived neurotrophic factor (BDNF). Endogenous brain-derived neurotrophic factor plays a central role in glutamate receptor-mediated survival pathways and provides a basis for the use of this neurotrophic factor to help the neurons to survive stressful conditions such as stroke and neurodegenerative disorders. Studies in rat stroke models have shown that intravenous brain-derived neurotrophic factor reduces infarct size and is a potent stimulator of adult neurogenesis. Animal experimental studies have demonstrated a critical role for brain-derived neurotrophic factor in rehabilitation-induced recovery after stroke, suggesting that treatment to enhance brain-derived neurotrophic factor is a promising therapy for promoting recovery of function in stroke patients. Brain-derived neurotrophic factor administration has been shown to lead to better functional outcome, oligodendrogenesis, remyelination, and fiber connectivity in rats subjected to subcortical damage in ischemic stroke (18).
Circulating concentrations of brain-derived neurotrophic factor are lowered in the acute phase of ischemic stroke, and these are associated with poor long-term functional outcome of these patients (19). However, further studies are required to confirm these associations and to assess the predictive value of brain-derived neurotrophic factor in stroke outcomes. In poststroke patients with hemiparesis, the combination of rehabilitation with low-frequency rTMS seems to improve motor function in the affected limb by activating processing of brain-derived neurotrophic factor (16). Therapy increased brain-derived neurotrophic factor, but not proBDNF levels in the serum.
Potential mechanisms of the neuroprotective role of brain-derived neurotrophic factor in focal cerebral ischemia include the following:
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• Protective effects against glutamate toxicity
• Attenuation of apoptosis
• Counter-regulation of Bax and Bcl-2 proteins within the ischemic penumbra
Cerebral dopamine neurotrophic factor (CDNF). CDNF is a member of MANF family of neurotrophic factors with neurotrophic effect in dopaminergic neurons. It has neuroprotective effects on cerebral ischemia in experimental animals and the oxygen glucose depletion cell model, which may occur through endoplasmic reticulum stress (MANF) pathways (22).
Neurotrophin 3 (NT3). Randomized, blinded preclinical trials have shown that intramuscular injection of adeno-associated viral vector (AAV1) encoding human NT3 (hNT3) promotes sensory and locomotor recovery in adult as well as old rats, even when treatment is initiated 24 hours after stroke (05). NT3-induced sensorimotor recovery is considered to occur through neuroplasticity and not neuroprotection. Phase 1 and 2 human clinical trials for other indications have demonstrated safety of repeated, peripherally administered high doses of recombinant NT3, easing the way for NT3 as a therapy for stroke.
Glial cell line-derived neurotrophic factor (GDNF). However, glial cell line-derived neurotrophic factor is a large molecule that cannot cross the blood-brain barrier. Topical application of glial cell line-derived neurotrophic factor greatly reduced the infarct size and brain edema at 24 hours of continuous middle cerebral artery occlusion in rats. Pretreatment of animals with glial cell line-derived neurotrophic factor by adenoviral gene therapy before the subsequent transient middle cerebral artery occlusion can reduce infract volume without affecting regional cerebral blood flow. PEP-1 protein transduction can deliver protein cargo across the cell membrane and the blood-brain barrier. A fusion protein, PEP-1-GDNF, given intravenously immediately after reperfusion of 90 minutes transient middle cerebral artery occlusion in rats significantly reduced the infarct volume and improved behavioral function (12).
Nerve growth factor. Nerve growth factor has a neuroprotective action after cerebral ischemia, but clinical application is hindered by lack of a suitable method for systemic delivery of nerve growth factor into the ischemic region of the brain. Engineered exosomes with rabies viral glycoprotein (RVG) peptide on the surface for targeting neuron can be loaded with nerve growth factor for delivery into ischemic cortex after systemic administration (21). The preparation is stable and functions efficiently for a long time in vivo with a burst release of encapsulated nerve growth factor protein translated from the delivered mRNA, which has a neuroprotective function.
Insulin-like growth factor-1. Preclinical studies have demonstrated that insulin-like growth factor-1 can protect against neuronal and glial cell degeneration in animal models of stroke. The higher insulin-like growth factor-1 levels observed in stroke patients with better outcome suggest a possible neuroprotective role of insulin-like growth factor-1. It is administered intracerebroventricularly because it does not cross the blood-brain barrier. However, this invasive method of administration is not practical for the large number of individuals who require treatment for stroke. To bypass the blood-brain barrier, it can be delivered to the brain directly from the nasal cavity following intranasal administration. This noninvasive, simple, and cost-effective method is a potential treatment for stroke.
Neuregulin-1. This neurotrophic factor is expressed throughout the immature and adult central nervous system and influences the migration of a variety of cell types in developing brain. Elevated levels of neuregulin-1 transcript are found in the adult brain after injury. Rats pretreated with neuregulin-1 protein and subjected to cerebral ischemia later have more recovery of motor performance and less cerebral infarction than untreated rats. Neuregulin-1 reduces ischemia/reperfusion injury, indicating that it may act as an endogenous neuroprotective factor against stroke. Neuroprotection possibly occurs through an inhibition of apoptotic pathways. In a rat model of middle cerebral artery occlusion/reperfusion, neuregulin-1 beta exerted neuroprotective effects by inhibiting the expression of calpain 1, p35/p25, and p-Tau after cerebral ischemia–reperfusion injury (22a). Neuregulin-1-based therapy has a potential for neuroprotection in patients with cerebral ischemia.
Granulocyte colony-stimulating factor (G-CSF). G-CSF is approved for the treatment of neutropenia associated with chemotherapy. Although not a neurotrophic factor, it has been shown to have neuroprotective effect as it activates astrocytes to release neurotrophic factors. G-CSF also mobilizes intrinsic stem cells. G-CSF reduces infarct volume and improves functional outcome after transient focal cerebral ischemia in mouse models. No clinical trials of G-CSF have been performed in cerebrovascular disorders, and it is difficult to determine the factors that improve functional outcome in stroke patients.
Granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF is a hematopoietic cytokine responsible for the proliferation, differentiation, and maturation of cells of the myeloid lineage. It is now considered to have a neuroprotective effect and is available as novel drug candidates for stroke. No clinical trial has been reported so far.
Use of neurotrophic factors as neuroprotective agents. Various studies of neurotrophic factors have shown that they have a neuroprotective action and that they enhance cellular systems involved in the maintenance of Ca2+ homeostasis and free radical metabolism. Several other compounds that mimic the action of neurotrophic factors by activating signal transduction cascades involving tyrosine phosphorylation have proven to be beneficial in animal studies of ischemic brain injury, and they provide opportunities for the development of therapeutic approaches for ischemic stroke. The most promising neurotrophic factor for neuroprotection in cerebral ischemia currently is the brain-derived neurotrophic factor.
Another promising neurotrophic factor is glial cell line-derived neurotrophic factor. Glial cell line-derived neurotrophic factor can protect the cerebral hemispheres from damage induced by middle cerebral arterial ligation. Such neuroprotective effects are mediated through specific glial cell line-derived neurotrophic factor receptor alpha-1. Exogenous administration of glial cell line-derived neurotrophic factor by transferring its gene via an adenoviral vector reduces the infarct size in middle cerebral artery occlusion in rats. Further studies are required to determine the relative potency of various neurotrophic factors, therapeutic windows, modes of administration, and doses.
Production of intrinsic neurotrophic factors in stroke by activation of astrocytes. Following cerebral ischemia, astrocytes become highly reactive and can exert neuroprotection through the release of neurotrophic factors and clearance of neurotoxic glutamate. A study has shown that neuron-derived 17β-estradiol (E2) is neuroprotective and critical for induction of reactive astrocytes and their ability to produce astrocyte-derived neurotrophic factors, BDNF and IGF-1, as well as the glutamate transporter, GLT-1 after ischemic brain damage (13). According to the authors of this study, beneficial effects of neuronal-derived E2 are due, at least in part, to suppression of neuronal FGF2 signaling, which is a known suppressor of astrocyte activation. These findings suggest that neuronal-derived E2 is neuroprotective after ischemic brain injury via a mechanism that involves suppression of neuronal FGF2 signaling, thereby facilitating astrocyte activation.
Neuroprotection by repair of blood-brain barrier. In a rat model of ischemia-reperfusion injury, pigment epithelium-derived factor and epidermal growth factor administered intravenously for 4 hours following reperfusion, reduced lesion volume expansion after reperfusion and persistently modulated blood-brain barrier permeability (17). In rats subjected to 1 hour of focal cerebral ischemia, early treatment with pigment epithelium-derived factor or epidermal growth factor resulted in lesion size reduction and blood-brain barrier stabilization, but glial activation was not influenced by treatment (15). The findings of ischemia-induced cellular reactions within the neurovascular unit might help in developing treatments to address the transition from injury towards regeneration.
Method of administration of neurotrophic factors for cerebral ischemia. In relation to treatment of cerebral ischemia, neurotrophic factors may be administered systemically by intravenous injection, or they can be delivered directly to the brain by intraventricular, intracerebral, or regional intraarterial injection. They can also be introduced into the CSF circulation by intrathecal injection. To improve delivery, brain-derived neurotrophic factor has been fused with laminin, an extracellular matrix that is highly expressed in the ischemic region after cerebral ischemia, and its administration in a rat model of middle cerebral artery occlusion resulted in a reduction of infarct volume as well as improvement in neurologic functional outcome (07). Transplantation of genetically modified stem cells that secrete neurotrophic factors into the brain is an effective method of delivery for the treatment of stroke.
Neuroprotective effect of other agents mediated via neurotrophic factors. There is experimental evidence that dexamethasone exerts a protective effect in experimental cerebral ischemia by modulating the expression of brain-derived neurotrophic factor and may provide specific trophic support for various neurons in the central nervous system.
Cerebrolysin. This is a neuropeptide preparation that mimics the action of endogenous neurotrophic factors on brain protection and repair. Studies in animal models of stroke have shown that cerebrolysin stabilizes the structural integrity of cells by inhibition of calpain, reduces the number of apoptotic cells after ischemic lesion, decreases infarct volume, induces restorative processes, and promotes functional recovery (14). Although cerebrolysin is not approved for use in the USA, it is used clinically in over 50 countries worldwide. Subgroup analyses of clinical trials shows that efficacy of cerebrolysin in stroke patients increases with stroke severity. Other controlled studies have shown that cerebrolysin can be safely used in combination with thrombolysis. It has been tested not only for neuroprotection but also for its neurorecovery potential and shows efficacy in patients with moderate to severe stroke (03). A prospective study has shown that the combination of cerebrolysin with classical rehabilitation techniques, including physiotherapy, improves recovery in post-stroke patients more than any of the methods used alone (20). However, a review of literature has concluded that cerebrolysin therapy can potentially play a major role in the treatment of many neurologic diseases, but much remains to be elucidated about the efficacy of this treatment specifically for stroke, and more robust clinical data are needed to reach a consensus to define its therapeutic role (06).
Role of neurotrophic factors in action of stem cell therapy for cerebral ischemia. Brain-derived neurotrophic factor and nerve growth factor have been shown to increase significantly in the ischemic boundary zone in rat stroke models following treatment with intravenous bone marrow stromal cells and correlated with recovery of function.
Stem cells may exert their beneficial effects by generating neurotrophic substances in the poststroke brain. Improvement of tissue damage and functional deficits following transplantation of adipose tissue-derived stem cell in animal stroke models is attributed to secretion of neurotrophic factors, particularly insulin-like growth factor-1.
Transplantation of human neural progenitor cells following stroke has been shown to enhance structural plasticity of the affected brain, and the underlying mechanisms include secretion of neurotrophic factors such as vascular endothelial growth factor (01). Mesenchymal stem cells (MSCs) can secrete various neurotrophic factors that stimulate neurite outgrowth and protect neurons against brain ischemic injury (10). Intracranial transplantation of human adipose-derived MSCs promotes the expression of neurotrophic factors and neural tissue repair in rats with cerebral ischemia-reperfusion injury (11). MSCs that are genetically modified to produce neurotrophic factors also reduce ischemic damage in animal stroke models. Therefore, a combination of growth factors with stem cells would have synergistic action and may be more effective than either treatment alone.
Neurotrophic factors also enhance migration of stem cells into the scaffolds used for repair of brain damage due to chronic ischemia in experimental animals. This effect has potential future application for regeneration of the damaged brain in chronic stroke patients.
A conditioned medium derived from adult neural progenitor cells, when administered intravenously to mice with focal cerebral ischemia, led to long-term neuroprotection that was associated with increased brain glial cell line–derived neurotrophic factor and vascular endothelial growth factor concentrations, resulting in increased neurogenesis and angiogenesis (04). The sustained neuroprotection and neurologic recovery suggest that cell transplantation may be replaced with secreted factors for therapeutic benefit. Bone marrow-derived MSCs promote recovery in a rat stroke model of distal middle cerebral artery occlusion, and neurotrophic factors IGF-1 and BDNF, which are mainly derived from transplanted MSCs as well as host microglia or macrophages, contribute to the therapeutic effects (09).
Clinical trials of neurotrophic factors for stroke. As of August 2021, the U.S. Government lists 42 clinical trials of neurotrophic factors for stroke and can be accessed at www.ClinicalTrials.gov.
In the past, clinical trials of neurotrophic factors for stroke have failed. Most of the current clinical trials are for the role of neurotrophic factors in other methods of treatment or neurotrophic factors as biomarkers. Only 2 clinical trials deal with administration of neurotrophic factors for stroke. One phase 4 trial is intranasal administration of nerve growth factor in ischemic stroke, but the current status is not known (NCT03686163). The other trials explore the use of hematopoietic growth factor in brain injury due to cerebral ischemia as well as other disorders (NCT02018406). Further experimental studies and refinements of methods of administration will improve the prospects of future clinical trials.
Concluding remarks and future. Despite promising results shown in animal models, critical evidence required for clinical application is inadequate. Further studies are required to elucidate optimal timing, dosing, and mode of administration, as well as possible combinations of neurotrophic factors with current management strategies.