Description

Pathomechanisms of damage to the nervous system as a basis for neuroprotection. An understanding of pathomechanisms of brain damage as well as normal neuroprotective mechanisms in the brain provide a basis for applications of neuroprotection.

Neuroprotective mechanisms in the normal brain. The glymphatic system, analogous to the lymphatic system of the rest of the body, consists of perivascular tunnels, formed by astroglial cells, to promote efficient elimination of waste products of cerebral metabolism from the CNS as well as to facilitates brain-wide distribution of several compounds, including glucose, lipids, amino acids, growth factors, and neuromodulators. The glymphatic system has a neuroprotective action, but it functions mainly during sleep and is mostly inactive during wakefulness. In sleep deprivation, the active process of the glymphatic system does not have time to perform that function, so toxins can build up, and the effects may manifest in cognitive abilities, behavior, and judgment. Glymphatic system is also impaired in early stages of Alzheimer disease leading to amyloid beta deposits. The action of glymphatic system, which is mainly mediated by water channel protein aquaporin 4 expressed in astrocytes, is impaired after traumatic brain injury and contributes to the accumulation of soluble proteins with secondary neuroinflammation (42).

Pathomechanism of central nervous system injury as basis for neuroprotection. The word injury is used here in a broad sense and implies damage to the central nervous system from a variety of diseases both acute and chronic. Although the cascades of molecular events following cerebral ischemia and trauma may differ, some basic pathological processes such as excitotoxic injury are common. The pharmacological strategies are aimed at 1 or more of these mechanisms. Some of these strategies are specific for conditions such as cerebral ischemia, whereas others are applicable to several disorders including ischemia, trauma, and neurodegeneration.

Intrinsic neuroprotective factors. Several protective mechanisms come into effect after injury to the nervous system. These protective mechanisms include neuroprotective gene expression and antioxidants such as superoxide dismutase. Gene-protein response to ischemia or injury includes c-fos/jun, hsp 70, transforming growth factor-beta1, nerve growth factor, and fibroblast growth factor. Within minutes of an insult, production of fos/jun increases, possibly triggered by the stimulation of glutamate N-methyl-D-aspartate receptors, changes in cyclic adenosine monophosphate, or increases in intracellular calcium. The effect may be translated into hsp, which acts as a molecular chaperone with a neuroprotective function. Up-regulation of genes for growth factors occurs within hours of an insult, but protein synthesis may take days to increase. Neurotrophic factors such as nerve growth factor and fibroblast growth factor have a neuroprotective function.

Autoimmune cells are generally viewed as a real threat as they can induce autoimmune diseases, but these cells can, if their levels are controlled, help to fight off debilitating neurodegenerative conditions as well as damage that results from trauma or stroke.

Neuroprotection in neuroinflammation. Pathogenesis of several neurologic disorders involves neuroinflammation, which is discussed in a MedLink article on this topic. Examples of these disorders include neurodegenerative diseases, traumatic CNS injury, and stroke. Antiinflammatory measures can be incorporated in neuroprotective approaches to these disorders.

Neuroprotective agents. A pharmacological classification of various neuroprotective agents is shown in Table 1. These approaches have been tested in animal experiments and clinical trials; however, some of these trials have not been successful. A few of the agents that are approved for nonneurologic disorders have been used empirically as neuroprotectives in clinical practice. Details of these compounds are described elsewhere (14), but some of these products will be described briefly in the following text.

Table 1. A Pharmacological Classification of Neuroprotective Agents

Adenosine analogs. Adenosine is an inhibitory neuromodulator and an endogenous neuroprotective agent. Stimulation of adenosine 1 receptors decreases excitatory amino acid transmission, and stimulation of adenosine 2 receptors inhibits platelet and neutrophil activation, thus, promoting vasodilatation. Adenosine triphosphates break down when ischemia produces adenosine, which effluxes out of the neurons. Propentofylline is a weak adenosine A1-receptor antagonist with neuroprotective effect.

Adrenergic receptor blockers. A study has shown that spontaneous waves of cortical spreading depolarization that are induced by acute focal ischemia cause a sharp increase of extracellular K+ that induces a long-lasting suppression of neural activity, resulting in secondary irreversible damage to the ischemic brain (24). The authors further reported that adrenergic receptor (AdR) antagonism by systemic adrenergic blockade accelerates normalization of extracellular K+, resulting in faster recovery of neural activity after stroke with the preservation of the water channel aquaporin-4 in astrocytes. Our observations suggest that AdR blockers promote cerebrospinal fluid exchange and rapid extracellular K+ clearance, representing a potent potential intervention for acute stroke.

Anesthetics. General anesthetics penetrate brain parenchyma and may prevent oxidative injury to neurons by inhibiting free radical generation due to slowing of cerebral utilization of oxygen and glucose. Antioxidant anesthetics, such as thiopental, are free radical scavengers. Anesthetics may also prevent the rise of extracellular glutamate concentration and inhibit the activation of excitatory glutamatergic receptors that aggravate oxidative stress after cerebral ischemia. Ketamine, a NMDA blocker, has been shown to be neuroprotective both in vivo and in vitro. As an anesthetic, ketamine is a neuroprotective in stroke, neurotrauma, subarachnoid hemorrhage, and status epilepticus (02).

Antidepressants. Antidepressants are used routinely in the management of neurodegenerative disorders. Several preclinical studies, using a variety of antidepressants, have shown neuroprotective effect but there is a paucity of clinical evidence. In studies on rats, a single dose of fluoxetine provides long-lasting protection against MDMA-induced loss of serotonin transporter and this neuroprotection is detectable in vivo by 4-18F-ADAM micro-PET (22). A study has reviewed the limitations of preclinical data and proposed an agenda for research on translational clinical neuroprotection (19).

Antiepileptic drugs. Some of the currently approved antiepileptic drugs also happen to have a neuroprotective effect, which has been demonstrated in animal models of hypoxia and ischemia. However, the prevention of epileptogenesis by an antiepileptic drug has yet to be demonstrated in clinical trials.

Antiinflammatory agents. Several antiinflammatory agents have been investigated for their neuroprotective effect. Nonsteroidal antiinflammatory drugs were reported to reduce the development of dementia in elderly subjects. Prostaglandin E2 may in some instances contribute toward excitotoxicity, and the inhibition of synthesis of this prostaglandin may in part explain the neuroprotective properties of COX-2 inhibitors, which act as neuroprotectives in vivo by suppressing toxic actions of microglia and macrophages. One antiinflammatory agent that has been used and investigated extensively as a neuroprotective is methylprednisolone, but the beneficial effect of methylprednisolone may be due to free radical scavenging. Methylprednisolone is a synthetic glucocorticoid used extensively in clinical trials as a high-dose neuroprotective agent in patients with spinal cord and brain injury. Despite the controversy, this approach is still used in clinical practice.

Apoptosis inhibitors. Apoptosis is inherently programmed cell death, distinct from cell necrosis. Apoptosis mediates cell deletion in tissue homeostasis, embryological development, and in pathological conditions such as cerebral infarction and neurodegenerative diseases. During conditions of degeneration, the cascade of events believed to cause cell death is initiated by an increase in synaptic glutamate levels, which results in an overstimulation of postsynaptic glutamate receptors. Consequently, a dramatic increase of intracellular calcium occurs, which leads to the formation of free radicals, such as nitric oxide. The excitatory amino acids, N-methyl-D-aspartate and glutamic acid, believed to cause many neurodegenerative diseases, induce apoptosis in neurons. Oxidative stress can also lead to apoptosis. Therapeutic approaches to block apoptosis include:

Beta lactam antibiotics. One of these, ceftriaxone, is a glutamate transporter, which prevents glutamate neurotoxicity by removing glutamate from nerves. Ceftriaxone is neuroprotective in vitro when used in models of ischemic injury and motor neuron degeneration, both based in part on glutamate toxicity. The antibiotic is currently approved by the FDA and used to treat bacterial infections in the brain. The neuroprotective effects, discovered in experiments in the laboratory, are unrelated to its ability to kill bacteria.

Caspase inhibitors. Apoptosis can be suppressed by agents that inhibit caspase activity. Caspase inhibitors are peptides resembling the cleavage site of known caspase substrates. Apart from naturally-occurring caspase inhibitors, pharmacological caspase inhibitors are either reversible or irreversible. Several caspase inhibitors are being evaluated for neuroprotective effects in animal models of cerebral ischemia and various neurodegenerative diseases.

Calpain inhibitors. Calpain was discovered more than 30 years ago as a Ca 2+ activated protease. Calpain activity increases in traumatic brain injury, spinal cord injury, cerebral ischemia, and Alzheimer disease. Calpain proteolysis represents a later component of a pathway mediating apoptosis initiated by excitotoxicity and elevated Ca 2+ levels. This provides an advantage for calpain inhibitors over drugs with conventional targets such as ion channels blockers and glutamate antagonists.

Poly (ADP-ribose) polymerase inhibitors. Poly (ADP-ribose) polymerase is an energy-intensive enzyme involved in the repair of damaged DNA. Nitric oxide generation can cause excessive activation of poly (ADP-ribose) polymerase, which rapidly leads to a complete depletion of a cell's energy levels, resulting in cell death. Poly (ADP-ribose) polymerase activation appears to be 1 of the final steps in the neurotoxicity cascade, which results in neuronal cell death in stroke and neurodegenerative disorders. Thus, the inhibition of poly (ADP-ribose) polymerase offers a unique approach to the development of novel neuroprotective agents. Novel poly (ADP-ribose) polymerase inhibitors are being developed to become neuroprotective agents during stroke and other disorders.

Free radical scavengers. Free radicals form during normal respiration and oxidation. During oxidation, a molecule transfers 1 or more electrons to another. Stable molecules usually have matched pairs of protons and electrons, whereas free radicals have unpaired electrons and tend to be highly reactive, oxidizing agents. Damage to cells caused by free radicals includes protein oxidation, DNA strand destruction, an increase of intracellular calcium, activation of damaging proteases and nucleases, and peroxidation of cellular membrane lipids. Furthermore, such intracellular damage can lead to the formation of prostaglandins, interferons, trinitrofluorenone-alpha, and other tissue-damaging mediators, each of which can lead to disease if overproduced in response to the oxidative stress. Free radicals have been linked to numerous human diseases including neurodegenerative diseases and ischemia reperfusion injury resulting from stroke. The level of free radicals is higher in neurodegenerative diseases than normal physiological condition as it is related to oxidative damages to neuronal cells, and use of antioxidants contributes to neuroprotection by decreasing oxidative stress (33).

The damaging effect of free radicals is controlled to some extent by the antioxidant defense systems and by cellular repair mechanisms of the body. Enzymes such as superoxide dismutase, catalase, glutathione peroxidase, and vitamins such as tocopherol, ascorbate, and beta carotene quench radical chain reactions. Many of these agents have been investigated as potential therapeutic agents. Unfortunately, most studies testing naturally occurring antioxidants result in disappointing results. Generally, natural antioxidants must be produced on site within the cell to be effective in disease prevention. Several of the synthesized antioxidant compounds have undergone clinical trials. Some have failed to demonstrate efficacy as neuroprotectives in stroke and head injury, whereas others are still undergoing clinical trials (or have completed the trials, although none have been approved as yet). However, some of the drugs used clinically are known to have neuroprotective effect (eg, dopamine agonists used for the treatment of Parkinson disease).

Neuroleptic drugs such as chlorpromazine, prochlorperazine, metoclopramide, methotrimeprazine, and haloperidol may be able to exert antioxidant or pro-oxidant actions in vivo, in vitro, or both. All neuroleptic drugs, except haloperidol, are powerful scavengers of hydroxyl radicals reacting at an almost diffusion-controlled rate with little reaction with the superoxide radical. Chlorpromazine shows some ability to inhibit iron ion-dependent hydroxyl radical formation. Chlorpromazine, methotrimeprazine, promethazine, and prochlorperazine are also powerful inhibitors of iron ion-dependent liposomal lipid peroxidation, scavengers of organic peroxyl radicals, and inhibitors of hem protein and hydrogen, peroxidations of arachidonic acid.

Gamma amino butyric acid agonists. Compounds that enhance gamma-amino-butyric acid A receptor responses have neuroprotective action. Ganaxolone belongs to a novel class of neuroactive steroids called epalons. It is a structural derivative of progesterone, but is devoid of hormonal activity. Epalons specifically modulate gamma-amino-butyric-acid A receptors in the central nervous system. Ganaxolone is in clinical phase II for epilepsy but its neuroprotective function was demonstrated in a rodent model of cerebral hemorrhage.

Cell therapy. Applications of cell therapy are described in the article on cell therapy for neurologic disorders. Stem cell transplantation is in clinical trials, including those for stroke and neurodegenerative disorders. Application of genetically engineered cells to release therapeutic substances in the brain is a form of gene therapy.

Gene therapy. It is feasible to protect neurons from a variety of insults using viral vectors carrying genes for neurotrophic factors. This approach has been used in Alzheimer disease and Parkinson disease.

Glutamate antagonists. Glutamate is the principal excitatory neurotransmitter in the central nervous system. Transmitter glutamate is taken and stored in synaptic vesicles by an ATP-dependent process and, after depolarization, is released into the synaptic cleft in a calcium dependent fashion. Glutamate is rapidly cleared from the extracellular space by sodium-dependent transporters located on glial and neuronal cell membranes.

Neuronal death, following excessive glutamate-mediated excitation, often referred to as excitotoxicity, is a critical feature of acute cerebral infarction. Increase in synaptic glutamate levels mediates apoptosis in neurodegenerative disorders. Overactivity of the excitatory amino acid neurotransmission is also associated with traumatic injuries of the central nervous system.

The largest segment of the pharmaceutical neuroprotective drug development activity focuses on the antiexcitotoxicity approach. Stroke was the first therapeutic area to be investigated. Some of the agents still under investigation are the following:

Memantine. Memantine, approved for the treatment of Alzheimer disease, is a noncompetitive, low-affinity N-methyl-D-aspartate antagonist with high-voltage dependency and fast receptor kinetics. It has antiexcitotoxic action and neuroprotective properties. Phase III trials for vascular dementia have been completed. A double-blind, placebo-controlled phase II human clinical trial of memantine for AIDS-related dementia is in progress.

Magnesium. The magnesium ion blocks the N-methyl-D-aspartate channel in a voltage-dependent fashion, but electrophysiologically, extracellular magnesium behaves as a noncompetitive N-methyl-D-aspartate antagonist. Neuroprotective effect of intra-arterial magnesium sulfate has been shown in animal models of reversible focal cerebral ischemia. This compound may be useful for limiting infarction if given intra-arterially before induction of reversible ischemia during cerebrovascular surgery. The advantages of using magnesium include availability, safety, and low cost. A randomized, placebo-controlled, double-blind pilot trial of intravenous magnesium sulfate in acute stroke patients showed an improved early outcome in magnesium sulfate recipients. A phase III trial is currently in progress.

Ceresine. Ceresine is a novel NR2B subunit antagonist of the N-methyl-D-aspartate receptor and has been shown to lower serum and cerebrospinal fluid lactate concentrations immediately after intravenous injection in various animal stroke models. Additionally, ceresine slows the development of cytotoxic edema and decreases the infarct volume after ischemic stroke. Ceresine is well tolerated in healthy volunteers, as well as in critically ill stroke patients. It ranks highly among the current neuroprotection candidates for clinical trials. Furthermore, ceresine maintains an excellent safety record in both animals and phase II studies in conscious, moderate head injury patients, suggesting that it will be highly effective.

NAALADase inhibitors. These compounds act as antiglutamate agents by inhibition of the enzyme NAALADase (N-Acetylated-Alpha-Linked-Acidic-Dipeptidase) at presynaptic levels and protect against neurodegeneration in a number of in vitro and in vivo animal models. A newly described NAALADase inhibitor, 2-PMPA (2-phosphonomethyl pentanedioic acid), protects against ischemic injury in a neuronal culture model of stroke and in rats after transient middle cerebral artery occlusion (34). Neuroprotection has been shown in a rodent model of 1-methyl-4phenyl-1, 2, 3, 6-tetra-hydropyridine Parkinson disease as well. NAALADase inhibitors do not interact with postsynaptic glutamate receptors, as such, they are devoid of the behavioral toxicity associated with postsynaptic glutamate antagonists.

Glutathione. The cytoprotective effects of glutathione play an important role in neuroprotection from neurotoxicity. Glutathione protects against the selective toxicity of 2-chloropropionic acids in the cerebellum by modulating brain thiol status. This subject is complex because certain chemicals may be converted to neurotoxicants following conjugation with glutathione such as in case of (±)-3, 4-methylenedioxyamphetamine-mediated serotonergic neurotoxicity.

Osteopontin. Osteopontin is an extracellular phosphoprotein involved in several biological functions, including inflammation, cell migration, and antiapoptotic processes. Osteopontin protects cortical neurons in culture against cell death due to oxygen and glucose deprivation and is a potential neuroprotectant.

Hyperbaric oxygen therapy. Hyperbaric oxygen therapy describes the therapeutic use of oxygen under greater atmospheric pressure. Its neuroprotective effects include relieving hypoxia, improving the microcirculation, relieving cerebral edema, protecting the partially damaged tissue, and preventing the secondary effects of infarction (13).

Hypothermia. Experimental studies have shown that a combination of intra-ischemic and postischemic hypothermia provides potent and persistent neuroprotection against temporary focal ischemia in experimental animals. Mechanisms of neuroprotective action of mild hypothermia are as follows:

Mild hypothermia (33 to 34 degrees Celsius) reduces cerebral edema and intracranial pressure in patients with extensive cerebral infarction. Hypothermia, following cerebral ischemia, delays the onset of ischemic histopathological alterations. Therefore, hypothermia may extend the therapeutic window for pharmacological neuroprotection, and may play an important part as an adjunctive treatment for thrombolytic therapy.

The first use of hypothermia, although mostly discarded, was in head injury. However, clinical trials resumed in the 1990s and showed that patients with severe traumatic brain injury had faster neurologic recovery compared to the course of patients treated under normothermic conditions. In patients who have been fully resuscitated after cardiac arrest due to ventricular fibrillation, therapeutic mild hypothermia increases the rate of a favorable neurologic outcome and mortality. Mild therapeutic hypothermia improves short-term neurologic recovery and survival in patients resuscitated from cardiac arrest of presumed cardiac origin.

Ion channel blockers. The following are used for neuroprotection:

Calcium channel blockers. Ca 2+ overload may play a critical role in ischemia, as well as various degenerative disorders of the nervous system. The development of improved strategies to prevent Ca 2+ overload is of importance for neuroprotection. Voltage-sensitive calcium channel blockers, particularly those that block excitatory amino acid release (eg, glutamate) have been shown to be neuroprotective following experimental ischemic insult to the brain. The success of Ca 2+ antagonists in cardiovascular indications has encouraged research of their therapeutic potential in cerebrovascular conditions.

Nimodipine is the most extensively studied calcium channel blocker for stroke. It has both cerebrovasodilatory and neuroprotective effects at doses that have little or no effect on peripheral circulation. Nimodipine is effective in delayed ischemic deficits of subarachnoid hemorrhage and when given within the first few hours after stroke. However, the compound has been relatively ineffective in clinical trials of stroke when given 13 hours to 24 hours after onset of symptoms. Further development of nimodipine for the indication of ischemic stroke has been discontinued after reaching phase III, although the product is available in the market of subarachnoid hemorrhage treatment.

Na+ channel blockers. In cerebral ischemia, energy demand exceeds the supply. Neuroprotection may be achieved by not only restoring cerebral perfusion, but also by decreasing the metabolic demands of the neural tissues. Down regulation of the Na+ channels, another effective way of reducing energy demand, allows a large part of the energy consumed by the brain to be used for the maintenance of ion gradients across the cellular membranes. A key component linking energy metabolism to transmembrane ion transport is the Na+/K+-ATPase, an enzyme that uses adenosine triphosphate to transport Na+ back out of the cell and K+ back into the cell, thus, restoring the ionic gradient across the cellular membrane. Down regulation of the Na+ channels in ischemia reduces the Na+ influx into the brain cell resulting in energy preservation. This also prevents the intrinsic neurotoxicity of the acute Na+ influx as well as the linked Ca 2+ influx.

Various pharmacological agents that exert their neuroprotective effect by down regulating Na+ channels are lamotrigine, phenytoin, and riluzole. Fosphenytoin is in clinical trials to evaluate its neuroprotective effect. Carbamazepine, 1 of the older anticonvulsants, provides for a neuroprotective effect that has been demonstrated in animal models of stroke, but has not been clinically evaluated. Lamotrigine inhibits the presynaptic release of glutamate by blockade of voltage dependent Na+ channels. Lamotrigine has been effective as demonstrated in cardiac arrest-induced global cerebral ischemia with reperfusion in rats.

Leukocyte adhesion inhibitors. Leukocytes are involved in central nervous system ischemic injury by means of the following mechanisms: direct microvascular occlusion after endothelial, basement membrane adhesion, transendothelial migration of leukocytes with secondary central nervous system tissue infiltration, and neuronal cytotoxic injury. Adhesion of leukocytes to microvascular endothelium is essential for initiation of any of these mechanisms, mechanisms that potentiate reperfusion injury as well.

Monoclonal antibodies. A natural human recombinant monoclonal antibody, rHIgM12 can promote neurite outgrowth in vitro by overriding the neurite outgrowth inhibition of myelin by binding with high affinity to neuronal PSA-NCAM (polysialic acid-neural cell adhesion molecule) and gangliosides (17). This neurite outgrowth is accompanied by increased α-tubulin tyrosination and decreased acetylation, which occurs after treatment with rHIgM12. rHIgM12 is effective in murine models of human multiple sclerosis and amyotrophic lateral sclerosis by improving axon survival and neurologic function. Thus, rHIgM12 has great potential as a therapeutic molecule in central nervous system disorders characterized by neuronal loss and axonal transection including multiple sclerosis.

Neuroglobin. Neuroglobin, the counterpart of hemoglobin in brain tissues, is a monomeric globin with high affinity for oxygen and can be detected in the CSF. Its affinity for oxygen and expression in cerebral neurons suggest a role in neuronal responses to hypoxia/ischemia. Neuroglobin has been demonstrated to be neuroprotective against stroke and related neurologic disorders.

Neuroimmunophilins. The immunosuppressants tacrolimus (FK-506) and cyclosporin are in clinical use for the treatment of allograft rejection following organ transplantation. Reports describe antiischemic activity of FK-506 and cyclosporin, which is explained by the antiinflammatory action. Other explanations are inhibition of nitric oxide synthase activity, suppression of apoptosis, and suppression of calcium-dependent signal transduction pathway that promotes interleukin-2 gene transcription in helper T-cells and direct neuronal action.

Cyclosporin protects the brain by interfering with the energy-producing mitochondria, keeping them viable during stroke by blocking mitochondrial cytochrome C release and apoptotic cell death. Additionally, it protects the brain by blocking calcineurin enzyme activity and nitric oxide production. Cyclosporin has a potential neuroprotective role in stroke, brain, spinal cord injury, and neurodegenerative diseases.

FK-506 and cyclosporin mediate their antiischemic activity by inhibiting apoptosis. The related immunosuppressant rapamycin, unlike FK-506, does not have a neuroprotective effect. FK-506 has been shown to ameliorate skilled motor deficits produced by middle cerebral artery occlusion in rats.

Neurotrophic factors. Neurotrophic factors are described in separate articles in MedLink. Endogenous BDNF 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. Several experimental studies in rat models of stroke have shown neuroprotective effect of glial cell line-derived neurotrophic factor (GDNF) delivered by local administration or by cell and gene therapies. However, glial cell line-derived neurotrophic factor is a large molecule that cannot cross the BBB.

Cerebral dopamine neurotrophic factor (CDNF) has a neurotrophic effect in dopaminergic neurons. It has a neuroprotective effect on cerebral ischemia in experimental animals and in the oxygen glucose depletion cell model, which may occur through endoplasmic reticulum stress pathways (44).

Nitric oxide. Nitric oxide is a neurotransmitter involved in normal brain functioning. In the nervous system, nitric oxide can be both a neurotransmitter and a neurotoxin. Nitric oxide differs from most conventional neurotransmitters in that it is not stored in synaptic vesicles. Therefore, synthesis of nitric oxide must be capable of rapid modulation. During pathologic conditions such as stroke, calcium overload causes prolonged activation of the enzyme nitric oxide synthetase. This results in an excess amount of nitric oxide release causing neural damage. The involvement of nitric oxide during stroke has been demonstrated in various animal models of stroke. Nonselective nitric oxide synthetase inhibitors may worsen stroke damage, presumably through inhibition of endothelial nitric oxide synthetase, which decreases cerebral blood flow. Selective neuronal nitric oxide synthetase inhibitors are being developed as neuroprotectives for neurologic disorders. Disorders in which nitric oxide and other neurotoxins play a significant part in eliciting neuronal death include: ischemic cerebrovascular disorders, traumatic brain and spinal cord injuries, and neurodegenerative disorders.

Clinical applications

Several approaches to neuroprotection, aimed at various steps in the cascade of events leading to damage or death of neurons, have been successful. Translation of this research into clinical practice remains largely unfilled. Despite several failed clinical trials, efforts continue in terms of developing therapies aimed at ameliorating the process of traumatic and ischemic damage.

The place of neuroprotection in the therapy of neurologic diseases, which involves damage to the central nervous system, is shown in Table 2:

Table 2. Place of Neuroprotection in 3 Phases of Management of Disorders of the Central Nervous System

An example of the application of the scheme shown in Table 2 is stroke, in which prevention may be instituted in high-risk procedures. The treatment of the cause of stroke, such as a thrombus, may be thrombolytic therapy, which should be supplemented with neuroprotective therapy. In chronic neurodegenerative diseases such as Parkinson disease, neuroprotection may continue in all 3 phases of the disease. Neuroprotective therapy has been used or investigated in diseases shown in Table 3 and is discussed in the following text:

Table 3. Diseases Where Neuroprotective Therapy Has Been Used or Investigated

Cerebrovascular disorders. Neuroprotection in stroke is the subject of several preclinical studies and clinical trials. The large number of failed clinical trials has raised a reasonable concern in approaching the future development of neuroprotective therapies in stroke. Citicoline is a membrane lipid precursor that reduces the progression of cerebral infarction. Produced endogenously, citicoline serves as a choline donor in the metabolic pathways for biosynthesis of acetylcholine and neuronal membrane phospholipids, chiefly phosphatidylcholine. Citicoline is approved only in Japan and Europe. A randomized, phase III study in the U.S. involved patients with acute ischemic stroke who presented within 24 hours of symptom onset and were randomized to receive oral citicoline or placebo for 6 weeks. Citicoline was found to be safe but ineffective in improving the outcome of patients with acute ischemic stroke. However, an individual patient data pooling analysis of clinical trials showed that treatment with oral citicoline within the first 24 hours after onset in patients with moderate to severe stroke increases the probability of complete recovery at 3 months.

Imatinib, a tyrosine kinase inhibitor approved for the treatment of some leukemias and other cancers, has been shown to restore blood-brain barrier integrity and reduce infarct size, hemorrhagic transformation, and cerebral edema in stroke models treated with tissue plasminogen activator. A phase 2 randomized trial showed that imatinib is safe and may reduce neurologic disability in ischemic stroke patients treated with intravenous thrombolysis (40). A confirmatory randomized trial is ongoing.

Statins are approved for hypercholesterolemia and prevention of cardiovascular disease. A metaanalysis of clinical trials has confirmed the class effect of statins in preventing major cerebrovascular events (26). Mechanisms of neuroprotective effects of statins include the following:

It is logical to consider combination of neuroprotection with thrombolysis for stroke. Immediate neuroprotection may enlarge the time window for thrombolysis. Moreover, following thrombolysis, reperfusion will improve the access of neuroprotective agents to reach the damaged brain. This concept is supported by studies in animal models but it has not as yet been rigorously tested in human clinical trials. The use of neuroprotective agents can prolong the therapeutic time windows in acute stroke prior to reperfusion and prevent reperfusion injury (35).

Hundreds of drugs have been tested in animal models of stroke, but none of them have been approved for clinical use as neuroprotective agents. The failure of most neuroprotective drugs in clinical trials has been due to inadequate preclinical testing and flawed clinical development programs. Discovery of new compounds with multiple mechanisms of action or setting of new standards for a rigorous preclinical testing led to only 2 new compounds, uric acid and nerinetide, to be tested for clinical efficacy in randomized controlled trials, where all patients had to receive reperfusion therapies, either intravenous thrombolysis and/or mechanical thrombectomy (05). In addition, otaplimastat, 3K3A-activated protein C, intra-arterial verapamil, and intra-arterial hypothermia were also assessed in combination with reperfusion therapy. Some of these compounds yielded promising results.

Pioglitazone, an approved oral antidiabetic medication, reduces the risk of stroke by directly targeting insulin resistance, a condition in which the body does not efficiently use the insulin it produces to control blood glucose levels. In a clinical trial involving patients without diabetes who had insulin resistance along with a recent history of ischemic stroke or transient ischemic attacks, the risk of stroke was lower among patients who received pioglitazone than among those who received placebo (16).

Arteriovenous malformations. A study has identified activating KRAS mutations in tissue samples of arteriovenous malformations of the brain and proposed that these malformations develop as a result of KRAS-induced activation of the MAPK (mitogen-activated protein kinase)-ERK (extracellular signal-regulated kinase) pathway in brain endothelial cells (27). These processes can be reversed by inhibition of MAPK-ERK signaling. Because no direct pharmacologic inhibitors of KRAS are available, small-molecule MEK inhibitors, which are in clinical use as anticancer drugs, may be tested in clinical trials to inhibit growth and hemorrhages in arteriovenous malformations of the brain.

Cerebral cavernous malformations. These malformations have an annual hemorrhage rate of 2.5%. Previous hemorrhage has shown to be the most significant hemorrhagic risk factor, whereas age, sex, location, size, and multiplicity are less relevant. Hyperpermeability of cerebral cavernous malformation vessels occurs via the inactivation of ccm1, 2, or 3 genes and the subsequent increase in Rho-associated coiled-coil-forming kinase (ROCK). In an experimental study, murine cerebral cavernous malformation models carrying the knockout allele for the tumor suppressor gene Trp53 or Msh2 were randomized to receive simvastatin, Fasudil (a specific ROCK inhibitor), or placebo (32). Both drugs significantly decreased chronic hemorrhage in cerebral cavernous malformation lesions, but Fasudil was more effective than simvastatin in improving survival and blunting the development of mature cerebral cavernous malformation lesions. This study shows the potential of ROCK inhibitors and the clinical testing of commonly used statin agents in cerebral cavernous malformations.

Neuroprotection in cerebral circulatory arrest. Brain damage usually occurs 3 to 6 minutes after total deprivation of blood flow and oxygen such as occurs in cardiac arrest, but the period may be longer with neuroprotective agents such as hypothermia or neuroprotective drugs. Brain damage is permanent after 10 minutes, and by 20 minutes, it extends to the deeper brain structures, eg, the brain stem. Neuroprotective strategies for prolonging the period of survival of the brain during induced circulatory arrest during surgery are discussed elsewhere (14).

An experimental study has described restoration and maintenance of microcirculation and molecular and cellular functions of the intact pig brain under ex-vivo normothermic conditions up to 4 hours after death and by using an extracorporeal pulsatile-perfusion system with an acellular cytoprotective perfusion fluid containing hemoglobin to promote recovery from anoxia, reduce reperfusion injury, prevent edema, and support the energy requirements of the brain (39).

Traumatic brain injury. Novel neuroprotective interventions include glutamate antagonists, calcium channel blockers, antioxidants, corticosteroids, and hypothermia. The pharmaceutical approaches have not been approved, although a few have completed phase III. Some of the trials were discontinued due to lack of efficacy or adverse effects. Infusions of magnesium within 8 hours of moderate or severe traumatic brain injury have failed to show neuroprotective effect.

Brain injury during neurosurgical procedures is inevitable where incision and retraction of the brain are involved. It may be worthwhile to institute preoperative neuroprotective treatment to reduce the effects of surgical trauma on the brain. Induction of anesthesia with propofol reduces cerebral blood flow but maintains coupling with cerebral metabolic rate for oxygen and decreases intracranial pressure, allowing optimal intraoperative conditions during neurosurgical operations for traumatic brain injury.

The complexity of sequelae of traumatic brain injury requires a multifaceted approach. In addition to the investigation of drugs for neuroprotective effect in traumatic brain injury, new technologies based on cell and gene therapies, biomarkers, and nanobiotechnology are being employed for the integration of neuroprotection with neuroregeneration (12).

Omega-3 fatty acids have been demonstrated to have neuroprotective effect in traumatic brain injury when given prior to the injury (21). This is the basis for consideration of Omega-3 fatty acids as nutritional prophylactic neuroprotective agents considered for those at risk or high exposure to brain impacts such as the athletes and soldiers.

Although the benefits of specific neuroprotective agents in traumatic brain injury have not been proven in randomized controlled trials, clinical outcomes of the patients have been improved by meticulous monitoring and prevention as well as limitation of secondary insults in the early phases after injury (36).

Neuroinflammation following traumatic brain injury may contribute to neurodegeneration, but administration of antiinflammatory drugs and immunosuppression have not been effective for the treatment of traumatic brain injury. Neuroinflammatory response seems to play a beneficial role in the acute phase of traumatic brain injury by setting the stage for regeneration. Therefore, modulation of inflammation for a favorable outcome rather than global suppression is recommended in acute traumatic brain injury (29).

In a large animal model of traumatic brain injury and hemorrhagic shock, early treatment with a single dose of mesenchymal stem cell-derived exosomes significantly attenuated brain swelling and lesion size, decreased levels of blood-based cerebral biomarkers, and improved blood-brain barrier integrity (41).

Acute spinal cord injury. The role of neuroprotection in acute spinal cord injury aims to prevent secondary injury, which is a series of deleterious events that promote progressive tissue damage and ischemia following the initial mechanical insult. Various therapeutic agents that have been investigated for this purpose include antioxidants, stem cells, corticosteroids, gene therapy, and hyperbaric oxygen. The North American Clinical Trials Network Consortium has selected 5 agents for conducting preclinical neuroprotection/pharmacotherapy trials–riluzole, glyburide, magnesium sulfate, nimodipine, and minocycline–because of their translational potential for management of spinal cord injuries (37).

A double-blind, randomized, placebo-controlled, dose-optimization, phase-2 study of minocycline for spinal cord injury demonstrated its safety but not efficacy (04). A commentary on this study pointed out the need for evaluation of drug therapy according to specific categories of spinal cord injury patients (23). Patients with incomplete spinal injury may show better recovery than those with complete injury where the cord is anatomically intact. It is obvious that those with structural disruption of continuity of spinal cord are unlikely to respond to drug therapy.

Cognitive decline with aging. The topic is discussed in the MedLink article on this topic. Neuroprotective measures, both pharmacological and nonpharmacological, have been used to manage aging patients with decline of mental function that goes beyond healthy aging and, in some cases, may be due to early stages of neurodegenerative disorders such as Alzheimer disease. Physical and mental exercises are beneficial, and the role of calorie restriction and intermittent fasting is being investigated. Epidemiologic data show that excessive energy intake, particularly in aging, increases the risks of neurodegenerative disorders. Preclinical studies have shown that the onset and progression of neurodegenerative disorders can be delayed in animal models by alternate-day fasting. A randomized trial of calorie restriction in healthy adult individuals showed a slightly positive effect on working memory and opens new possibilities to prevent and treat cognitive deficits (20). However, data from controlled trials of intermittent fasting in persons at risk for or affected by a neurodegenerative disorder are lacking. Ideally, an intervention would be initiated early in the disease process and continued long enough, eg, a 1-year study, to detect a disease-modifying effect of the intervention (06).

Parkinson disease. Current therapies in Parkinson disease are aimed mainly at symptomatic relief. Current understanding of the molecular pathophysiology of the disease provides the possibility of neuroprotective therapy to halt or reverse the process of the disease. The neurodegenerative process in Parkinson disease may involve a cascade of events including oxidative stress, mitochondrial dysfunction, and excitotoxicity leading to cell death, which offers targets for neuroprotective therapy. The slow evolution of the neurodegenerative process, which begins before the symptomatic phase, provides an opportunity for slowing the rate of progression of the disease to be fostered. An additional reason for considering neuroprotective therapy in Parkinson disease is the possibility that levodopa and its metabolites may kill cells by causing lipid peroxidation, membrane disruption, and damage to the mitochondrial respiratory apparatus. The categories of neuroprotective therapies in Parkinson disease are as follows:

Of the above list, only anti-Parkinson drugs with antioxidant effects, dopamine agonists, and deep brain stimulation are approved for clinical use; the others are in clinical trials. Dopaminergic drugs smoothen out dopaminergic tone. They could possibly interrupt the vicious cycle of dopamine deficiency that leads to disinhibition of the subthalamic nucleus and glutamate-induced excitotoxicity. This provides a rationale for the use of dopamine agonists and selective N-methyl-D-aspartate receptor antagonists to block glutamate effects. Although preclinical evidence from laboratory models suggests potential neuroprotective benefits, the antioxidant, antiapoptotic, antiexcitotoxic, immunomodulatory, and neurotrophic agents have not shown clear benefit in human studies. Obstacles to the development of a neuroprotective therapy in Parkinson disease include uncertainty as to the precise cause of cell death in this disease. New developments in understanding the cause of the disease and in clinical trial methods are expected to address these problems. Clinical trials designed to show neuroprotection (selegiline, amantadine, dopamine agonists) demonstrate that with the drugs available, neuroprotection and neurorescue have to start as early as possible in the early phase of Parkinson disease. No such benefit was seen in patients with a late start of a neuroprotective therapeutic strategy. Some dopaminergic drugs have entered clinical trials for neuroprotection, and a few have produced positive results according to the endpoint selected. One lesson for neuroprotection that emerges from these trials is simply to treat early rather than delay.

Atremorine is a natural levodopa donor for Parkinson disease, with effects on dopaminergic neurons. In a study on patients with plasma dopamine levels below 20 pg/mL at diagnosis, a single dose of Atremorine induced a significant increase in plasma dopamine levels 1 hour after administration in nearly all patients (03). Atremorine-induced dopamine response was pharmacogenotype-specific and varied according to pharmacogenetic profile of each patient. Genetic variants in pathogenic genes, metabolic genes, and genes involved in the detoxification processes affected the response of dopamine to Atremorine in a genotype-specific manner. Atremorine or any of its bioactive components can cross the blood-brain barrier and improve brain function as well as motor activity. Atremorine is a selective neuroprotective agent for dopaminergic neurons with prophylactic and therapeutic potential in Parkinson disease. Safinamide inhibits MAO-B activity as well as glutamate release (25). It combines both dopaminergic and nondopaminergic actions that may add a new dimension to treatment of Parkinson disease as an adjunct to current drugs (31). It is in phase III clinical trials, and there are also efforts to demonstrate the cognition-improving and disease-modifying features of safinamide.

Calcium has a role in the normal physiological function of alpha-synuclein by binding to it at its C terminus and increases its lipid-binding capacity leading to alpha-synuclein aggregation and toxicity related to Parkinson disease (18). An understanding of the role of interaction of calcium with alpha-synuclein in pathological processes may help in the development of new treatments for Parkinson disease, and the potential of calcium channel blockers should be explored as neuroprotective agents.

Several attempts have been made in the past to treat Parkinson disease with neurotrophic factors. Most of these were not successful mainly because of difficulty in delivering these factors to the site of action in the brain. A phase II clinical trial is in progress in patients with Parkinson disease using stereotactic intrastriatal injection of an adeno-associated viral vector carrying the gene for neurturin, a member of the same family as glial cell line derived neurotrophic factor.

Surgical interventions can also inhibit neuronal firing in the subthalamic nucleus. Deep brain stimulation is approved for use in Parkinson disease, but the underlying mechanisms of action are uncertain. Deep brain stimulation can induce plastic changes in glial-neuronal interaction networks of the brain and may increase delta-opioid receptor activity in astrocytes to confer neuroprotection (08). In a computational model of the corticobasal ganglia-thalamocortical loop in normal and parkinsonian conditions, high-frequency stimulation was shown to regularize firing pattern and restore the neural activity in downstream nuclei (30).

Alzheimer disease. Multiple mechanisms are involved in the pathogenesis of Alzheimer disease. The factor most relevant to neuroprotection by currently available agents is oxidative stress. The therapies are based on cholinergic augmentation but target 1 of the several disturbances in Alzheimer disease. Therapies relevant to neuroprotection include:

Extended combination therapy of memantine with an acetylcholinesterase inhibitor has been shown to have a neuroprotective effect in Alzheimer disease by slowing cognitive and functional decline compared with acetylcholinesterase inhibitor monotherapy or no treatment (01).

Huntington disease. Knowledge of the genetic defect in patients with Huntington disease coupled with the availability of genetic testing have enabled the detection of individuals at risk for Huntington disease prior to the onset of symptoms. Symptomatic treatments are available, but no cure. It is important to find neuroprotection strategies in these persons. Discovery of the Huntington disease gene and its product, huntingtin, has improved understanding of the disease lesion and opened new approaches to curative treatments. However, the mechanism by which the genetic defect (unstable trinucleotide repeat) leads to neuronal degeneration is not known. Glutamate-mediated excitotoxicity and abnormalities of mitochondrial energy production are implicated and may lead to production of free radicals. The goal for treatment of Huntington disease is to develop neuroprotective therapies that can delay or prevent illness in those who are at genetic risk and can slow progression in those who are affected clinically. Preclinical discovery research in Huntington disease is identifying numerous targets along with options for modulating them, and some of these are proceeding into large-scale efficacy studies in early symptomatic subjects (10). Some of the approaches that fall under the category of neuroprotection are the following:

Antiglutamate and free radical scavengers. Controlled clinical trials of antiglutamate agents remacemide hydrochloride as well as riluzole and free radical scavenger coenzyme Q10 in patients with Huntington disease have shown some neuroprotective effect in slowing the clinical disability without improving functional capacity or other clinical features of illness.

Neurotrophic factors. The role of neurotrophic factors and methods of administration have been discussed in a separate article on neurotrophic factors in the treatment of neurodegenerative disorders.

Cell transplants. Preliminary results from fetal striatal cell transplantation studies in a few patients with Huntington disease are encouraging. Confirmation of these results in a larger group of patients is required.

Amyotrophic lateral sclerosis. Riluzole is considered to have neuroprotective effects in amyotrophic lateral sclerosis by blocking the repetitive discharge of sodium action potentials and increasing the threshold for generation of calcium spike. SR 57746A, a 5-hydroxytryptamine 1A agonist with neurotrophic-neuroprotective effects, is in phase III for amyotrophic lateral sclerosis. Homocysteine exerts multiple neurotoxic mechanisms that are relevant in the pathogenesis of amyotrophic lateral sclerosis, and reduction of homocysteine levels by methylcobalamin may be useful for modifying disease progression and possibly its onset as well. In preliminary studies in patients with amyotrophic lateral sclerosis who had higher median homocysteine levels compared to age- and sex-matched controls, a high dose of methylcobalamin was effective in improving compound motor action potentials (46). Currently, mecobalamin intramuscular injection is in phase II/III clinical trials for amyotrophic lateral sclerosis at multiple centers in Japan.

Epilepsy. Seizures may be a manifestation of various brain disorders, such as ischemia or traumatic brain injury, but prolonged seizures or status epilepticus may also lead to neuronal damage. These aspects are taken into consideration for neuroprotection. Some of the currently approved antiepileptic drugs also have a neuroprotective effect. The neuroprotective effect of antiepileptic drugs is classified according to their mechanism of action as follows:

Na+ channel blockers. Blockers include carbamazepine, fosphenytoin, lamotrigine, and phenytoin. Interactions with voltage-sensitive calcium channels include felbamate, lamotrigine, topiramate, and gabapentin.

Gamma amino butyric acid agonists. Agonists include barbiturates, benzodiazepines, valproic acid gabapentin, felbamate, topiramate, tiagabine, vigabatrin, and zonisamide.

Antiglutamate action. None of the established older drugs, however, have antiglutamate action, whereas the newer drugs such as felbamate and topiramate act by this mechanism. Among the antiepileptic drugs in development, the following have neuroprotective effect by reduction of glutamate-mediated excitation. Remacemide hydrochloride, a noncompetitive N-methyl-D-aspartate receptor blocker, is in phase III for refractory partial epilepsy. It is also under investigation for the treatment of Parkinson disease. ADCI, a N-methyl-D-aspartate receptor channel blocker, is in phase II clinical trials.

Multiple sclerosis. During the long-term course of multiple sclerosis, neuroinflammation evolves into neurodegeneration, and these 2 processes require different treatment strategies – immune modulation for the former and neuroprotection for the latter (38). Selective immunomodulating drugs are more likely to have a neuroprotective effect than immunosuppressant drugs, which control the acute exacerbation of the disease. Neuroprotection in multiple sclerosis is often tied in with the regeneration therapies that aim for remyelination.

Peripheral neuropathy. There are numerous causes of peripheral neuropathy. An example is given of the measures used for protection of nerves in diabetic neuropathy, which include the following:

Recombinant human erythropoietin has a neuroprotective and neurotrophic action in preventing and reversing nerve dysfunction in streptozotocin-induced diabetes in rats. Alterations in mechanical and thermal nociception are partially reversed by erythropoietin. This suggests that erythropoietin or its analogs may be useful in the treatment of diabetic neuropathy.

Mitochondrial disorders. In addition to involvement of the nervous system in inherited mitochondrial diseases, mitochondrial dysfunction has been reported in several neurodegenerative diseases, eg, Parkinson disease. Advances in our understanding of the mitochondrial regulating pathways have provided several promising approaches to neuroprotection, such as drugs to modulate mitochondrial biogenesis and free radical scavengers as well as dietary measures such as ketogenic diet (28).

Elevated neuroglobin level is associated with preserved mitochondrial function in neurodegenerative disorders, suggesting that it may play neuroprotective roles through mitochondria-mediated pathways, especially those involved in adenosine triphosphate production and generation, as well as scavenging of reactive oxygen species (43). Mitochondrial fragmentation, an early event during apoptosis, is implicated in the degeneration of dopamine neurons in Parkinson disease. Histone deacetylase inhibitors prevent mitochondrial fragmentation and provide neuroprotection in early stages of Parkinson disease cell models indicating a potential that they could form the basis for developing early treatment for Parkinson disease (45).

Neonatal encephalopathy. The role of hypoxia-ischemia in the etiology of neonatal encephalopathy is controversial, but inflammation is recognized as a predisposing factor for neurodisability. A systematic review of clinical trials has shown that intravenous magnesium significantly reduces the risk for cerebral palsy in preterm birth (11).

Therapeutic hypothermia is standard of care in developed countries, but its role in long-term management is questionable. However, hypothermia, melatonin, erythropoietin, and cannabinoids can supplement the endogenous response to hypoxia-ischemia to achieve its full neuroprotective potential (09). A randomized trial of high-dose erythropoietin treatment administered to extremely preterm infants from 24 hours after birth through 32 weeks of postmenstrual age did not result in a lower risk of severe neurodevelopmental impairment or death (15).