Experimental neuroprotection in ischemic stroke: a concise review · Experimental neuroprotection in ischemic stroke: a concise review Gary B. Rajah, MD, and Yuchuan Ding, MD, PhD

NEUROSURGICAL

FOCUS Neurosurg Focus 42 (4):E2, 2017

Acute ischemic stroke (AIS) resulted in 3.3 million deaths globally in 2013 according to Feigin et al.24 They estimated that there were close to 18 million

ischemic stroke survivors worldwide in 2013. The global stroke burden continues to increase.24 Recent innovations in the clinical stroke arena include advanced imaging for stroke diagnosis9 and endovascular mechanical thrombec-tomy for reperfusion treatment of AIS due to large ves-sel occlusion, which has been validated in 7 randomized controlled trials.5,6,9,28,38,53,62 These new techniques, as well as intravenous tissue plasminogen activator, have trans-formed emergent stroke care. To date, however, there have been few successful adjuvant neuroprotective therapies to aid in the treatment of acute stroke. Such therapies would target reductions in secondary injury to penumbra tissue, minimizing damage before and after reperfusion while promoting neural recovery and plasticity. In this paper we review upcoming preclinical neuroprotective therapies as well as their proposed mechanisms of action. In addition, we evaluate the existing clinical data on neuroprotection in stroke.

Clinically Assessed Neuroprotective Therapies

In this review we first focus on therapies tested in pa-tients in either a pilot study or a randomized controlled

design. Despite the many preclinical studies suggesting clinical efficacy, there is a paucity of clinically proven therapies. We have created tables (Tables 1–3) recapping the clinically tested therapies and discuss the more rele-vant therapies below. Recently, randomized trials for acute stroke and neuroprotection have included growth factors, hypothermia, minocycline, natalizumab, fingolimod, and uric acid.11 Common pathways targeted for neuroprotec-tion include free radical scavengers, excitotoxicity, im-mune modulation, and more.

Free Radical ScavengersFree radicals are molecules with a free unpaired elec-

tron, making them highly reactive and capable of chain reactivity. They can react with and damage proteins, nu-cleic acids, and lipids.66 They are present in low levels via normal cellular respiration, but during acute ischemic epi-sodes their levels can increase and have been postulated to contribute to cerebral edema.29 We summarize the studies on free radical scavenging therapies in Table 1.

Reduction of ExcitotoxicityExcitotoxicity is a type of neurotoxicity centered

around glutamate. During acute ischemia, the extracellu-lar concentration of glutamate rises quickly and stimulates N-methyl-d-aspartate receptor (NMDAR), which acts via calcium permeability. Excitotoxic cell death is triggered

ABBREVIATIONS AIS = acute ischemic stroke; MB = methylene blue; MCA = middle cerebral artery; NMDAR = N-methyl-d-aspartate receptor; Treg = regulatory T cell.SUBMITTED November 23, 2016. ACCEPTED January 26, 2017.INCLUDE WHEN CITING DOI: 10.3171/2017.1.FOCUS16497.

Experimental neuroprotection in ischemic stroke: a concise reviewGary B. Rajah, MD, and Yuchuan Ding, MD, PhD

Department of Neurosurgery, University Health Center, Wayne State University, Detroit Michigan

Acute ischemic stroke (AIS) is a leading cause of disability and death worldwide. To date, intravenous tissue plasmino-gen activator and mechanical thrombectomy have been standards of care for AIS. There have been many advances in diagnostic imaging and endovascular devices for AIS; however, most neuroprotective therapies seem to remain largely in the preclinical phase. While many neuroprotective therapies have been identified in experimental models, none are cur-rently used routinely to treat stroke patients. This review seeks to summarize clinical studies pertaining to neuroprotec-tion, as well as the different preclinical neuroprotective therapies, their presumed mechanisms of action, and their future applications in stroke patients.https://thejns.org/doi/abs/10.3171/2017.1.FOCUS16497KEY WORDS neuroprotection; acute ischemic stroke; free radical; excitotoxicity; immune response

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G. B. Rajah and Y. Ding

Neurosurg Focus Volume 42 • April 20172

via PTEN, cdk5, and DAPK1, among other downstream targets.42 We summarize the studies on excitotoxic block-ing therapies for neuroprotection in Table 2.

Immune ModulationThe immune response after ischemic stroke is multifac-

eted and initiates a cascade leading to secondary brain in-jury following the initial ictus as well as after reperfusion therapies. Gelderblom et al.27 reported increased microglia activation and an influx of macrophages, lymphocytes, and dendritic cells with increased antigen-presenting capabil-ity. The brain under normal conditions is able to maintain a relatively noninflammatory environment; however, once

the brain is damaged, inflammation is left less regulated.2 We summarize studies on immune modulation therapies in Table 3.

Other TherapiesThere are many ongoing clinical trials examining

statins, imatinib, dapsone, interleukin-1 receptor antago-nists, NA-1, hypothermia, lower limb tourniquet condi-tioning, insulin infusion, edaravone, and 3K3A-APC.11

American Heart Association Stroke Guidelines Related to Early Management and Neuroprotection

There are no Level 1 recommendations for neuropro-

TABLE 1. Clinical free radical scavenging therapies for neuroprotection in AIS

Authors & Year

Trial Name Agent MOA

No. of Patients in Study Benefit w/ Agent Details

Diener et al., 2008

SAINT NXY-059 Free radical trapping 5028 No clinical benefit Treated w/in 6 hrs of symptoms

Clark et al., 2001

Citicoline Membrane stabilizer/free radical scavenger

899 No clinical benefit Daily administration for 6 wks post-stroke

Yamaguchi et al., 1998

Ebselen Seleno-organic compound w/ antioxidant properties

300 Better outcome at 1 mo but not 3 mos; improvements in mBI

Oral administration × 2 wks post-stroke

Chamorro et al., 2014

URICO-ICTUS

Uric acid Antioxidant 411 No significant benefit, but 39% vs 33% (placebo) attained mRS score ≥2

Administered w/in 4.5 hrs w/ alteplase

mBI = modified Barthel Index; MOA = mechanism of action; mRS = modified Rankin Scale; SAINT = Stroke Acute Ischemic NXY Treatment; URICO-ICTUS = Efficacy Study of Combined Treatment with Uric Acid and r-TPA in Acute Ischemic Stroke.

TABLE 2. Clinical ion channel interaction/excitotoxicity blockade therapies for neuroprotection in AIS

Authors & Year Trial

Name Agent MOA


Wahlgren et al., 1999

CLASS Clomethiazole GABA A potentiator inducing membrane hyperpolarization

1360 No clinical benefit; in subgroup analysis, increased functional independence w/ large stroke

Administered w/in 12 hrs of stroke onset

Albers et al., 2001

Aptiganel hy-drochloride

Selective ligand for NMDAR 628 No clinical benefit & possibly harmful


Davis et al., 2000

Selfotel NMDAR antagonist 567 Stopped early given possible neurotoxic effects


Horn et al., 2001 VENUS Nimodipine Ca++ channel blocker 454 No beneficial effect Administered w/in 6 hrsLiu et al., 2009 Ginsenoside Ca++ channel blocker 199 Some benefit as determined by

15 day NIHSS14-day infusion

Saver et al., 201563

FAST-MAG

Magnesium Vasodilatory, neural & glial protective

1700 No clinical benefit at 90 days Administered w/in 2 hrs & a 24-hr infusion

Hill et al., 2012 NA-1 Inhibitor of postsynaptic density protein-95*

197 NA-1 group had fewer ischemic infarcts on DWI, FLAIR imag-ing 12–95 hrs postinfusion

Infusion at end of endo-vascular procedure

Ladurner et al., 2005

Cerebrolysin Purified brain protein made from enzymatic digestion of brain protein w/ neurotrophic/pro-tective properties†

146 Significant improvement in cognition as tested by Short Syndrome Test

Administered w/in 24 hrs, for 21 days

CLASS = Clomethiazole Acute Stroke Study; DWI = diffusion-weighted imaging; FAST-MAG = Field Administration of Stroke Therapy-Magnesium; GABA = γ-aminobutyric acid; NIHSS = National Institutes of Health Stroke Scale/Score; VENUS = Very Early Nimodipine Use in Stroke. * Postsynaptic density protein-95 is believed to regulate gating and surface expression of NMDA channels.45 † Orphan category added here for convenience.


Review of neuroprotection in ischemic stroke

Neurosurg Focus Volume 42 • April 2017 3

tective agents in AIS. The 2013 American Heart Associa-tion stroke guidelines state the following: continuation of statin therapy is reasonable, the utility of induced hypo-thermia in AIS is not well established, transcranial near-infrared laser therapy for AIS is not well established, and the utility of hyperbaric oxygen therapy is inconclusive and may be harmful in AIS, unless due to air embolism.36 The guidelines also state that no neuroprotective pharma-cological agents have demonstrated clinical efficacy and thus are not currently recommended.

HypothermiaHypothermia can target more than one aspect of sec-

ondary brain injury, including oxygen consumption and metabolic demand,15 enzymatic degradation, neurotrans-mitter uptake,7 membrane stabilization, and reduction in intracellular acidosis.32,55 In a small study, hypothermia post–middle cerebral artery (MCA) stroke has been shown to be tolerable, help control intracranial pressure, and pos-sibly lead to better neurological outcomes.67 Post–cardiac arrest neural protection has also been demonstrated, with 55% of patients who underwent cooling having good out-comes versus 39% in the normothermia group.32

Other neuroprotective strategies related to hypother-mia in the clinical phase include an endovascular device inserted into the vena cava for cooling to 33°C for 24 hours,18 which was used in the Cooling for Acute Ische-mic Brain Damage (COOL AID) study. This therapy was generally well tolerated, with trends toward less lesion growth on diffusion-weighted imaging. In a more recent pilot study, 26 patients underwent intraarterial endovascu-lar cooling within 8 hours of intraarterial recanalization.13 This therapy did reduce brain temperature by at least 2°C with no obvious complications. The Intravascular Cool-ing in the Treatment of Stroke (ICTuS) 2 trial results were recently published;50 however, only 120 patients of the in-

tended 1600 were enrolled before the study was stopped. A femoral venous catheter was used for cooling. Mortality was 15.9% in the hypothermia group versus 8.8% in the normothermia group.

Kasner et al.39 performed a randomized controlled trial to determine if 3900 mg of acetaminophen administered daily to afebrile patients with acute stroke could reduce core body temperature, promote modest hypothermia, and prevent hyperthermia; however, these authors concluded that these effects are likely to have no robust impact on outcome. What has been shown is that fever in the first 24 hours of AIS leads to a doubling of the odds of death in 1 month post-stroke.58 Thus, despite the benefit of hypother-mia on functional outcomes post–cardiac arrest,32 its use in AIS is still unproven.

Hyperbaric OxygenSince AIS occurs when the brain tissue oxygen supply

does not meet the demand, many have investigated hyper-baric oxygen therapy as a way to boost brain oxygen lev-els. In a 2014 meta-analysis, Bennett et al.4 examined 11 randomized controlled trials assessing hyperbaric oxygen in AIS. These authors found no good evidence to suggest that hyperbaric oxygen improves clinical outcomes in AIS.

Near-Infrared Laser TherapyLow-energy laser therapy has been used as a potential

neuroprotective strategy in AIS. The theorized mechanism of action involves photostimulation of mitochondrial chro-mophores increasing enzymatic production of adenosine triphosphate, with resultant ischemic tissue preservation.54 A pooled analysis of the NeuroThera Effectiveness and Safety Trials (NEST) 1 and 2,35 involving 778 patients, supported the likelihood that transcranial laser therapy was effective when initiated within 24 hours of AIS. The

TABLE 3. Clinical immune modulation/antiinflammatory therapies for neuroprotection in AIS

Authors & Year Trial Agent MOA


Zhu et al., 2015

Fingolimod Immune modulator* 47 Smaller lesion vol (10 vs 34 ml), less hemorrhage, & better NIHSS; 90-day mRS Score 0–1 in 73% vs 32%

Administered w/ alteplase w/in 4.5 hrs, for 3 days

Elkins et al., 2016

ACTION Natalizumab Monoclonal antibody against α4 integrin

161 More patients had mRS Score 0–1 at 30 & 90 days; however, no effect on infarct growth

Single dose 0–9 hrs from symptom onset

Kohler et al., 2013

Minocycline Inhibit microglial & T cell ac-tivation, decrease neural apoptosis, inhibit MMP-9

95 No clinical benefit Infusion w/in 24 hrs & every 12 hrs for total of 5 doses

Schäbitz et al., 2010

AXIS A Filgrastim Granulocyte colony-stimulat-ing factor

44 No significant outcome difference, but exploratory analysis revealed some benefit for DWI lesions >14 cm3

4 doses over 3 days w/in 12 hrs of symptoms

Emsley et al., 2005

Interleukin-1 receptor antagonist

Blocks interleukin-1–medi-ated cell death

34 Clinical outcomes at 3 mos better w/ antagonist, decreased C-reactive protein & neutrophils

w/in 6 hrs of symp-toms for 72 hrs

ACTION = Effect of Natalizumab on Infarct Volume in Acute Ischemic Stroke; AXIS = Treatment With AX200 for Acute Ischemic Stroke; MMP-9 = matrix metalloprotein-ase. * Fingolimod acts via immune modulation and sequesters lymphocytes via sphingosine-phosphate receptor internalization, but also reportedly is a ceramide synthase inhibitor and cannabinoid receptor antagonist.




authors concluded that their findings were promising but needed confirmation.

Stem Cell TherapyIn 2016 Hess et al.30 reported the results of their Phase 2

trial of MultiStem (adult, adherent stem cell product). One hundred twenty-six patients (National Institutes of Health Stroke Scale [NIHSS] Score 8–20) were randomized to receive a MultiStem infusion or placebo within 24–48 hours of symptom onset. While no significant benefit was identified, post hoc analysis did suggest early administra-tion may provide some benefit. MultiStem treatment was associated with lower rates of infection and pulmonary events, shorter hospitalizations, and a reduction in life-threatening events and death.

Preclinically Assessed Neuroprotective Therapies

Despite the many promising therapies examined in clinical studies, none has yet gained guideline recommen-dation for the treatment of AIS. Many preclinical studies have been completed or are underway, and we review a few promising ones here.

Neuroprotection strategies have been traditionally di-vided into different mechanistic varieties including com-pounds or therapies that combat oxidative stress, the im-mune response, and excitotoxicity. Some therapies can target more than one aspect of the secondary brain injury following stroke.

Chlorpromazine/PromethazineThe phenothiazine class of drugs, which includes chlor-

promazine and promethazine, have widely been used as neuroleptics but have also been shown to have depressive effects on the central nervous system through the inhibi-tion of carbohydrate oxidation, as well as vasodilatory and anti-shivering properties.47 The drugs at high concentra-tions can induce hypothermia in rats.73 Liu et al.47 exam-ined the combination of phenothiazine drugs with mild hypothermia in a rat intraluminal MCA filament model of stroke. Combination therapy reduced infarct volume and resulted in better long-term motor recovery than placebo, phenothiazine alone, or hypothermia alone. The authors concluded that phenothiazine drugs may enhance the neu-roprotective effects of hypothermia by reinforcing depres-sive central nervous system effects and thus achieving goal hypothermia faster. These drugs can inhibit mitochondrial dysfunction and decrease free radical production.

Caffeinol (caffeine and ethanol)Aronowski et al.,3 utilizing a rat model of common

carotid artery/left MCA infarct, treated different cohorts with combinations of ethanol, caffeine, and hypothermia. They concluded that low doses of caffeine, equivalent to 2–3 cups of coffee, and low doses of ethanol, equivalent to 1 cocktail, are highly neuroprotective and can reduce cortical infarct volumes and behavioral dysfunction after transient occlusion. However, daily exposure to ethanol eliminated the efficacy through tolerance, thought to be

related to NMDA and γ-aminobutyric acid (GABA) recep-tor changes. The caffeinol treatment was further improved with mild hypothermia. Recently, Cai et al.8 suggested that the neuroprotection mediated through mild hypothermia and ethanol was PKC-Akt-NOX mediated. A pilot study in humans did demonstrate the feasibility of the adminis-tration of both agents in AIS patients.51

Methylene BlueMethylene blue (MB) is a drug used to treat malaria,

methemoglobinemia, and cyanide poisoning.65 It is an FDA-grandfathered drug.37 Neuroprotective and memory-enhancing qualities have been demonstrated in neurode-generative disease.60 Methylene blue is thought to act as a renewable auto-oxidizer, redirecting electrons in the mitochondrial transport chain promoting ATP production and cell survival. In hypoxic conditions, MB becomes the oxidizer and sustains ATP production while lowering oxidative stress. Furthermore, MB enhances cytochrome c oxidase activity while bypassing complexes 1–3.37,75 It is also thought to augment HIF-1α activation and stabiliza-tion,61 increase cerebral blood flow to mismatched areas, and promote autophagy via p53-AMPK-TSC2-mTOR, while downregulating apoptosis via the p53-Bax-Bcl2-caspase 3 pathway in perfusion-diffusion mismatched tis-sue. Shen et al.68 found that MB in a 60-minute rat MCA model of stroke had no effect on the initial MRI-demon-strated lesion volume; however, at 2 days after stroke, the final infarct volumes increased in the vehicle group but decreased in the MB group, yielding a 30% overall in-farct difference. Pixel by pixel analysis showed that MB salvaged more core and penumbra pixels than the control treatment. Recently, the addition of normobaric hyperoxia to MB was found to further increase functional outcome and decrease infarct volume.59

RapamycinRapamycin is an immunosuppressant drug that targets

mTOR and its downstream pathways. mTOR has been im-plicated in regulating cell survival, proliferation, growth, metabolism, and autophagy. Recently, rapamycin was found to extend lifespan in a variety of animal models.25 Chauhan et al.,12 utilizing an MCA filament model in rats, found that rapamycin could significantly improve the in-farct area and apparent diffusion coefficient and improve motor impairment as compared with controls. It also re-versed the changes in malondialdehyde, glutathione, nitric oxide, and myeloperoxidase. When administered daily for 1 month before injury in diabetic rats, rapamycin was found to reduce ischemic brain damage via mTOR and ERK1/2 suppression.46 Not all rodent models of stroke have shown positive results with rapamycin; for example, the drug was found to increase infarct size and oxygen consumption in a rat model.14

Inflammatory/Stress Response and Immune ModulationThe inflammatory response in AIS has been called

a double-edged sword with early inflammation causing harm, but late inflammatory changes contributing to re-generation.69 Hu et al.34 found that macrophage subtype




shifted from early beneficial M2 microglia to M1 mi-croglia and suggested that new therapies should focus on adjusting the balance between good and bad subtypes. Recently, this shift from the M1 to the M2 phenotype in stroke was found to be mTOR mediated.43 Several anti-bodies targeting adhesion molecules designed to prevent leukotaxis have been produced in the last few years.74 Antibodies targeting anti-neutrophil markers have been shown to decrease myeloperoxidase injury, infarct vol-ume, and edema post-reperfusion.52 Intercellular adhe-sion molecules and integrins have also been targeted with varying degrees of success, and some very promising ex-perimental data have not translated into good clinical per-formances. One explanation is that rodents’ and humans’ inflammatory responses post-stroke involve different inte-grins and signaling responses.69 Remote ischemic precon-ditioning has been found to protect against focal ischemia and preserve neurological function via ameliorating post-occlusion reduction in CD3+/CD8+ T cells and CD3+/CD161a+ natural killer T cells. It induces interleukin-6 expression and tumor necrosis factor–α expression while increasing the number of noninflammatory monocytes (CD43+/CD172a+).49 Using a Cre-Lox system, De Mag-alhaes Fihlo et al.19 found that downregulation of insulin growth factor receptor-1 in cortical neurons also conferred neuroprotection and should be further investigated. This receptor is implicated in the stress response and hypoxic ischemic injury post-stroke.

Li et al.44 examined the therapeutic advantage of regu-latory T cells (Tregs) in 2 rodent models of stroke and in in vitro Treg-neutrophil cocultures. They found that sys-temic administration of Tregs out to 24 hours post–MCA occlusion resulted in a marked reduction in brain infarct and prolonged improvement in neurological function. This was achieved via decreased blood-brain barrier dis-ruption and decreased cerebral inflammation. The Tregs also decreased levels of matrix metalloproteinase-9. De-spite these promising results, however, many challenges remain, namely that Tregs constitute only 5%–10% of cir-culating T cells. Thus, they would need to be expanded either in vivo or in vitro and properly honed for cerebral tissue, and doing so carries its own challenges.71

Doeppner et al.21 examined lithium in a rat MCA oc-clusion model. They noted that when lithium was admin-istered within 6 hours of onset, reduced infarct volumes, edema, leukocyte infiltration, and microglia activation were present. They reported that lithium increased levels of miR-124, resulting in the degradation of RE1-silencing transcription factor and thus leading to postischemic neu-roplasticity. This effect was independent of glycogen syn-thase kinase 3b (GSK3b).

Finding New Ways to Use Old Therapies and Future Directions

Lastly, it is important to remember older, previously studied neuroprotective agents. As technology changes, the ability to target previously nonspecific drugs or to change the pharmacokinetics of previously rapidly cleared compounds changes. For example, in 2014 Gaudin et al.26 reported that the conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies

prolonged circulation and conferred neuroprotection in a rat stroke and spinal cord injury model. However, even though the delivery vehicle was improved, new data sug-gest a temporal effect of adenosine, namely that adenosine receptors support early excitotoxicity but then seem to attenuate the inflammatory response following.57 Regard-less of this temporal finding, as technology finds new ways to deliver or improve therapies, we must be open to reas-sessing older agents.

In a landmark paper on experimental neuroprotection related to AIS, published in 2006, O’Collins et al.56 sys-tematically reviewed 1026 experimental therapies in both focal and global ischemic animal models, with some stud-ies dating back to the 1950s. These authors compared 114 drugs used clinically to 912 drugs used experimentally and found the clinical therapies to be no more efficacious experimentally than the therapies tested experimentally only. Overall, 64% of drugs tested in the focal ischemic animal models were effective, versus 70% of drugs tested in the global ischemic animal models. Interestingly, no specific drug mechanism distinguished itself by superior efficacy in the focal ischemic animal models. The authors suggested that this finding may reflect the multifaceted nature of stroke or perhaps an incorrect understanding of secondary stroke injury. Thus, they urged rigorous pre-clinical testing and reporting of data, so that only the most efficacious therapies move forward to clinical phases.

While many of the studies predating 2005, as compared with those from the last 10 years, and the studies reviewed in the current paper target similar pathways, many of the immune modulating and immunotherapy techniques dis-cussed above have only recently been investigated. These studies, along with repeat validation of promising older drugs and therapies, will lay the foundation for stroke neu-roprotection moving forward. Many promising therapies are available, including hibernation-like therapy, immune modulation therapy, and near-infrared laser therapy. The disconnect between the beneficial effects of hypothermia for post–cardiac arrest global neuronal ischemia and the lack of efficacy for focal ischemia due to AIS requires better understanding. As mentioned in the O’Collins et al. study,56 perhaps researchers and clinicians need to stop compart-mentalizing AIS secondary injury and instead focus on the global picture. Targeting only one aspect of a multifaceted cascade will probably result in only mild benefits.

ConclusionsMany clinical and preclinical studies of neuroprotec-

tion in stroke have been completed; however, few studies have shown clinical benefit. It is imperative that relevant high-quality preclinical studies progress to randomized clinical trials. Neuroprotection in stroke remains very promising with many avenues for improvement and dis-covery, including trials of current and new therapies in combination. Given the benefits of hypothermia post–car-diac arrest, future studies should determine the exact role of therapeutic hypothermia in AIS, as well as the discon-nect between current post–cardiac arrest data and AIS data. Stem cell therapy and immunomodulation also ap-pear promising.




AcknowledgmentsThis work was partially supported by the WSU Neuro-

surgery Fund, American Heart Association Grant-in-Aid No. 14GRNT20460246, and Merit Review Award No. I01RX-001964-01 from the US Department of Veterans Affairs Rehabilitation R&D Service.

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DisclosuresThe authors report no conflict of interest concerning the materi-als or methods used in this study or the findings specified in this paper.

Author ContributionsConception and design: both authors. Analysis and interpreta-tion of data: Ding. Drafting the article: both authors. Critically revising the article: both authors. Reviewed submitted version of manuscript: both authors. Approved the final version of the manu-script on behalf of both authors: Ding. Study supervision: Ding.

CorrespondenceYuchuan Ding, Department of Neurosurgery, Lande Building, #48, 550 East Canfield, Detroit, MI 48201. email: [email protected].


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Experimental neuroprotection in ischemic stroke: a concise review · Experimental neuroprotection in ischemic stroke: a concise review Gary B. Rajah, MD, and Yuchuan Ding, MD, PhD