Ischemic preconditioning provides long-lasting neuroprotection against ischemic stroke: The role of Nrf2

Journal Pre-proof Ischemic preconditioning provides long-lasting neuroprotection against ischemic stroke: The role of Nrf2

Tuo Yang, Yang Sun, Qianqian Li, Senmiao Li, Yejie Shi, Rehana K. Leak, Jun Chen, Feng Zhang PII:

S0014-4886(19)30289-4

DOI:

https://doi.org/10.1016/j.expneurol.2019.113142

Reference:

YEXNR 113142

To appear in:

Experimental Neurology

Received date:

5 October 2019

Accepted date:

3 December 2019

Please cite this article as: T. Yang, Y. Sun, Q. Li, et al., Ischemic preconditioning provides long-lasting neuroprotection against ischemic stroke: The role of Nrf2, Experimental Neurology (2019), https://doi.org/10.1016/j.expneurol.2019.113142

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© 2019 Published by Elsevier.

Journal Pre-proof Title: Ischemic preconditioning provides long-lasting neuroprotection against ischemic stroke: the role of Nrf2

Authors: Tuo Yang, MD1 , Yang Sun, MD1 , Qianqian Li, MD1 , Senmiao Li, MD1 , Yejie Shi, MD,

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PhD1 , Rehana K. Leak, PhD2 , Jun Chen, MD, PhD1,3 , and Feng Zhang, MD, PhD1, From the Department of Neurology, Pittsburgh Institute of Brain Disorders and Recovery,

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University of Pittsburgh, Pittsburgh, PA (T.Y., Y.S., Q.L., S.L., Y.S., J.C., F.Z.); Division of

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Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA (R.K.L.); and Geriatric Research,

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Educational and Clinical Center, Veterans Affairs Pittsburgh Health Care System, PA (J.C.).

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Cover title: IPC offers long-term protection by activating Nrf2 Keywords: Stroke, neuroprotection, ischemic tolerance, electrophile, post-stroke cognitive

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impairment

Highlights:

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- Ischemic preconditioning provides long-lasting neuroprotection against sensorimotor and cognitive impairments as well as tissue loss after ischemic stroke; the protection is reduced in Nrf2 knockout mice.

- Mechanistically, preconditioning leads to sublethal generation of lipid electrophiles, which then mediate endogenous activation of the Nrf2 pathway. - Pharmacological activation of Nrf2 provides a similar degree of long-term protection in translational studies. - Nrf2 is a promising therapeutic target with enduring protective effects against ischemic stroke.

Journal Pre-proof Word count: 3887

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Total number of tables: 0

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Total number of figures: 6

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Journal Pre-proof Abstract: Background and Purpose: A major gap in the field of ischemic preconditioning (IPC) is whether or not long-lasting neuroprotection can be achieved. Moreover, the specific mechanisms underlying IPC and how they can be translated into the clinic remain uncertain. To fill these gaps, we tested the hypothesis that IPC exerts long- lasting structural and functional neuroprotection against ischemic stroke through the master gatekeeper of antioxidant defenses, nuclear factor erythroid 2-related factor 2 (Nrf2). We also tested whether the brain could be pharmaceutically preconditioned with a potent and blood-brain barrier-permeable Nrf2 activator, 2-cyano-3,12-

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dioxo-oleana-1,9(11)-dien-28-trifluoethyl amide (CDDO-TFEA).

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Methods: IPC was induced by transient middle cerebral artery occlusion (MCAO) for 12 minutes, and ischemic stroke was generated by MCAO for 60 minutes in wild-type (WT) or Nrf2

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knockout (KO) mice. Sensorimotor function, learning/memory skills, and brain tissue loss were

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measured up to 35 days after stroke. Primary rodent cortical neurons from wildtype (WT) and Nrf2 KO mice were subjected to lethal oxygen- glucose deprivation (OGD) or a brief OGD

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episode as a preconditioning (PC) stimulus before OGD. Cell viability/death, lipid electrophile generation, and Nrf2 activation were measured. CDDO-TFEA or its vehicle was administered in

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vivo for three consecutive days before MCAO. Tissue loss and neurological tests were performed 35 days after stroke.

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Results: IPC significantly reduced sensorimotor deficits, post-stroke cognitive impairments,

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and brain tissue loss, 35 days after MCAO in WT mice. These enduring protective effects of IPC were inhibited in Nrf2 KO mice. In neuronal cultures, PC also endowed primary neurons with ischemic tolerance against OGD- induced cell death, an effect that was abolished by loss of Nrf2 expression in KO neurons. PC induced the generation of low levels of lipid electrophiles and led to activation of the Nrf2 pathway. The mechanism underlying IPC may be translatable, as exogenous administration of the Nrf2 activator CDDO-TFEA significantly reduced neurological dysfunction and ischemic brain damage after MCAO. Conclusions: IPC provides long- lasting neuroprotection against ischemic brain injury and post-stroke cognitive dysfunction. Nrf2 activation plays a key role in this beneficial outcome and is a promising therapeutic target for the attenuation of ischemic brain injury.

Journal Pre-proof Introduction It has been known for many decades that ischemic preconditioning (IPC) of the brain is an effective approach to elicit ischemic tolerance against subsequent lethal stroke

1-3

. However,

whether IPC provides short- lasting or long- lasting neuroprotection is still debated. Two timeframes of IPC- mediated ischemic tolerance have been reported in the literature 3 : 1) a rapid timeframe that emerges within minutes and provides transient protection with a duration of several hours, and 2) a delayed timeframe that emerges within hours to days and confers a relatively longer protection period (> 7 days). Rapid preconditioning only provides short- lasting 4, 5

. Although several studies have reported that delayed

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protection that wanes within 1-3 days

6-9

, it

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non-ischemic preconditioning offers long-lasting protection against ischemic injuries

remains unclear if delayed ischemic preconditioning offers similarly long- lasting neuroprotection.

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As long- lasting protection would be more clinically meaningful, there is an urgent need to

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determine if IPC simply delays ischemic brain injury or reduces the magnitude of the injury in a persistent fashion.

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Post-stroke cognitive impairments affect up to one-third of stroke survivors

10

and are

becoming increasingly prevalent in the elderly population 11 . However, whether IPC can reduce

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post-stroke cognitive impairments is not known. As there is no treatment available against poststroke cognitive impairment and dementia, it is important to investigate if IPC can offer some

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degree of protection against post-stroke cognitive impairment.

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Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcription factor regulating cellular redox equilibrium by inducing the expression of myriad antioxidant genes previous study

17

2, 12-16

. In our

, we discovered that delayed IPC in mice activates the Nrf2 pathway, as

indicated by the upregulation of heme oxygenase 1 (HO-1) in neurons, astrocytes, and endothelial cells. Among the ten categories of Nrf2 activators

18

, the Michael reaction acceptors

are the only endogenous activators of this transcription factor. Michael reaction acceptors are electrophiles, and the majority are end products of lipid peroxidation, including 4- hydroxynonenal (4-HNE) from omega-6 fatty acids and 4- hydroxy-hexenal (4-HHE) from omega-3 fatty acids

19-22

.

Previous in vitro studies have demonstrated that preconditioning (PC) of astrocytes activated astrocytic Nrf2, which protected neurons against ischemic injury in astrocyte-neuron

Journal Pre-proof co-cultures 23, 24 ; however, it is unknown if direct PC of neurons can also prevent ischemic injury. It is well known that neurons are highly sensitive to oxidative stress and that PC is associated with sublethal oxidative stress

23, 25-27

. On this backdrop, we hypothesize that PC may lead to

mild oxidative stress in neurons via the generation of lipid electrophiles, followed by induction of self-defense mechanisms through electrophile- mediated Nrf2 activation and Nrf2- mediated transcription of Phase 2 antioxidant genes. To address the abovementioned gaps and explore the potential mechanisms behind IPC, we choose delayed IPC in the present study, as it confers more robust neuroprotection than rapid 28

and is dependent upon genetic reprogramming and de novo protein synthesis

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IPC

2, 25, 29

. We

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report that IPC can protect the brain against long-term sensorimotor and cognitive dysfunction and tissue loss after experimental ischemic stroke through the activation of Nrf2. Finally, to

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emphasize the importance of Nrf2 activation in long- lasting neuroprotection, we also performed

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a translational study with a potent and blood-brain barrier-permeable Nrf2 activator 2-cyano3,12-dioxo-oleana-1,9(11)-dien-28-trifluoethyl amide (CDDO-TFEA), which exerted similar

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robust and enduring neuroprotective effects.

Journal Pre-proof Methods Animals and blinding This study was designed in accordance with ARRIVE guidelines. All animal experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC) and carried out in accordance with the Stroke Treatment and Academic Roundtable (STAIR) guidelines and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult male C57BL/6 mice and Nrf2 knockout (KO) mice were purchased

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from The Jackson Laboratory (Bar Harbor, ME) and housed with a 12:12 hr light-dark cycle at 22-24°C. The mice were randomly assigned to sham surgery (Sham), Stroke, and IPC+Stroke

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using a lottery-drawing box. All the outcome assessments were performed by investigators

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blinded to the group assignments.

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Middle cerebral artery occlusion (MCAO)

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Male wild-type (WT) and Nrf2 KO mice underwent MCAO on the left side for 12 min to induce IPC 3, 30 , and 60 min to induce preclinical stroke 20, 31 . These procedures were followed by

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reperfusion for the indicated durations. The interval between IPC and stroke was three days. The effects of IPC on ischemic outcomes in female mice have been reported in our previous study

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MCAO was performed with standard, previously published procedures

17, 20, 31

17

.

. In brief,

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mice were anesthetized with 1.5% isoflurane in a 30% O 2 /70% N 2 O mixture under spontaneous breathing, and rectal temperature was maintained at 37.0 ± 0.5°C with a temperature-regulated heating pad. Mean arterial blood pressure was monitored with a tail-cuff. Under a surgical microscope, the left external, internal, and common carotid arteries were exposed through a midline neck incision. After coagulating and cutting the branches of the external carotid arteries, a 7-0 monofilament nylon suture with a silicone coat was inserted in the lumen of the external carotid artery and advanced to the origin of the middle cerebral artery via the internal carotid artery. The success of ischemia was confirmed by the measurement of regional cortical cerebral blood flow (rCBF) and the examination of neurological deficits

17

.

Journal Pre-proof Drug preparation and administration 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-trifluoethyl

amide

(CDDO-TFEA)

was

purchased from Cayman Chemical (Ann Arbor, MI) and prepared according to previous reports 32

. The mice received CDDO-TFEA (25 μmol/kg) or vehicle by gastric gavage in 200-μL

volumes for three consecutive days before MCAO.

Behavioral tests

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Sensorimotor functions were assessed with the Rotarod and adhesive removal tests

Morris water maze test for five weeks after stroke.

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Rotarod test

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beginning in the first week after stroke, and long-term cognitive function was assessed using the

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Mice were trained for three consecutive days before surgery and were examined up to 35 days after stroke. After the mice were placed on the rods (IITC Life Science Inc., Woodland

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Hills, CA), the rods began to rotate and accelerate to 40 rpm within 300 s. The maximal length of upright on the rods 17, 33, 34 .

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Adhesive removal test

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each trial was 300 s. Results are presented as the duration that the mice were able to remain

Mice were trained for five consecutive days before surgery and were tested up to 7 days

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after stroke. An adhesive paper patch (3 × 3 mm) was attached to the distal radial region of the right forelimb. The time to remove the patch from the forelimb was recorded

17, 20, 35

.

Morris water maze test Mice were first trained in the Morris water maze for three days before surgery, and the tests were initiated 31 days after stroke when motor deficits have largely recovered in this model. The pool was 110 cm in diameter (SDI, San Diego Instruments, San Diego, CA), whereas the platform was 11 cm in diameter and submerged 1 cm under the surface of opaque water filled with non-toxic paint. Mice underwent one hidden platform test per day for five days, during which each mouse was released from one of 4 quadrants and allowed to search for the hidden platform for 90 seconds (learning phase of the test). At the end of each trial, the mouse was

Journal Pre-proof placed on the platform or allowed to remain on the platform for 30 seconds. Prominent spatial cues were arranged around the room. Four trials were performed per day for five consecutive days, with the location of the platform kept constant. Spatial learning abilities were measured as the time (in seconds) for the animal to reach the submerged platform on each day (latency). At day 35, a single, 60-s probe trial was performed in which the platform was removed. The time spent in the target quadrant where the platform was previously located was recorded (memory phase of the test). Swim speed was also recorded to assess gross locomotor function

.

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Immunohistochemistry

20, 36

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Brains were sectioned in the coronal plane at 20 µm thickness and stained with anti-microtubule

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associated protein 2 (MAP2, 1:200; Santa Cruz Biotechnology, Dallas, TX). Tissue loss was calculated relative to the contralateral hemisphere by the following edema-corrected equation:

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(viable area of contralateral hemisphere - viable area of ipsilateral hemisphere) / viable area of the contralateral hemisphere). The viable areas in this equation were defined as the MAP220, 34

. Sections at the level of the dorsal hippocampus were immunostained

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immunoreactive zones

with an antibody against the specific neuronal marker NeuN (1:200, Abcam). For cell counting,

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the numbers of NeuN-positive CA1 neurons per mm2 were counted by NIH Image J.

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Primary neuronal cultures and oxygen-glucose deprivation (OGD)

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Primary cultures of the rodent cortical neurons were dissected from E16-18 fetuses and maintained in Neurobasal media supplemented with B27 (Gibco, ThermoFisher Scientific, Pittsburgh, PA), as previously described

20, 31, 37

. The experiments were performed 10-14 days

after seeding cells in culture plates. OGD was used to mimic in vitro ischemia with standard, previously published procedures

31

A brief duration of OGD (12 minutes) was used as a PC stimulus, prior to 1- h lethal OGD. The interval between PC and OGD was 16 h, and cells were returned to normal culture media and normal oxygenation for 24 h after OGD before harvest.

.

Journal Pre-proof Western blots and Western slot blots Total cell lysates from primary rat neurons were subjected to standard Western blot assays, as previously described

20, 31

. Protein concentrations from total neuronal lysates were determined

by Bradford protein assay (Bio-Rad, Hercules, CA). Equal amounts of proteins were subjected to electrophoresis, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA). Membranes were probed with antibodies recognizing HO-1 (1:1000, Enzo Life Science, Farmingdale, NY) and β-actin (1:3000, Sigma-Aldrich, St. Louis, MO). After incubation in secondary antibodies (Santa Cruz Biotechnology, Dallas, TX), membranes were

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incubated with chemiluminescent substrates (Pierce, ThermoFisher Scientific, Pittsburgh, PA)

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and developed with X-ray film. ImageJ software was used for gel analyses. For the Western slot blot assays, proteins were loaded directly onto PVDF membranes using

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a Bio-Dot Microfiltration apparatus (Bio-Rad, Hercules, CA). Anti-4-HNE (1:1000, R&D,

antibodies

17, 38

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Minneapolis, MN) and anti-tubulin (1:2000, Abcam, Cambridge, MA) were used as the primary . The remaining procedures were the same as described above for the Western

Cell death and viability

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blots.

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Cell death and viability were measured by the Live/Dead cell viability assay (Molecular Probes, ThermoFisher Scientific, Pittsburgh, PA), the lactate dehydrogenase (LDH) release assay

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(Pointe Scientific Inc., Canton, MI), and the methylthiazolyldiphenyl- tetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO). These assays were performed in triplicate, on at least three independent occasions.

The Live/Dead cell viability assay was performed according to the manufacturer’s instructions (Molecular Probes, Eugene, OR), as previously described 17, 20 . In this assay, red dots (fluorescent ethidium homodimer-1) represent dead cells with compromised membranes, whereas green dots (fluorescent membrane-permeant calcein AM) represent live cells. The numbers of red dots were expressed as a function of red plus green dots and were presented as “cell death percentages”. For cell counting, three random fields were photographed per well by a

Journal Pre-proof blinded observer. Four to six wells per condition per experiment were selected, and the experiments were repeated on at least three independent occasions. For the MTT assay, cells were incubated with 0.5 mg/mL MTT solution for 40 min, when purple precipitates became visible. After two washes, cells were lysed with dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO), in which the purple precipitates dissolved. Cell viabilities were then determined by absorbance at 595 nm, and data were expressed relative to the control group.

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Extracellular LDH release by damaged cells was measured by an LDH detection kit (Pointe Scientific, Inc., Canton, MI), according to the manufacturer’s instructions. All data were

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expressed as the percentage LDH release relative to the control group.

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Statistical analyses

The results are presented as the mean ± SD for the in vivo studies and mean ± SEM for in

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vitro studies. The difference between means was assessed by Student’s t-test (single comparisons) or by the appropriate analysis of variance (ANOVA, for multiple comparisons). GraphPad Prism

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software (version 7.0, La Jolla, CA, USA) was used for statistical analyses. A p value  0.05

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Results

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was deemed statistically significant.

IPC protects against long-term cognitive defects and brain tissue loss To determine if IPC offers long-term neuroprotection in mice, we performed the Rotarod test up to 5 weeks after stroke. As shown in Fig. 1A, IPC significantly improved sensorimotor function in this assay, for at least five weeks post- injury. To assess post-stroke cognitive function, we performed the Morris water maze tests in the fifth week after stroke. As shown in Fig. 1B-D, IPC significantly improved learning and memory capacities after stroke. Notably, swim speeds did not differ across groups, suggesting that the water maze results could not be attributed to differences in gross motor skills (Supplementary Fig. 1A). Immediately following the memory test at 35 d post-stroke, the mice were sacrificed, and the brains were harvested for

Journal Pre-proof immunostaining with the neuronal marker MAP2 to assess brain tissue loss. As shown in Fig. 1E, IPC significantly reduced tissue loss compared to the stroke group, indicating a long-term neuroprotective effect of IPC. Hippocampal neurons play an important role in learning and memory, particularly those in the CA1 fields. To investigate whether stroke and IPC affect CA1 neuron viability, we counted neurons immunostained for the specific neuronal marker NeuN. As shown in Supplementary Fig. 2, there were no significant differences between control and preconditioned groups in NeuN+ cell counts after MCAO. We also performed TUNEL staining to detect dead cells. While TUNEL-positive signals were visible at the rim of the infarction in

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the cortex, little signals could be detected in the hippocampi of both groups (Data not shown).

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Nrf2 plays a key role in IPC-mediated long-term neuroprotection in vivo Next, we tested the hypothesis that Nrf2 is essential for IPC-mediated neuroprotection. For

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these mechanistic experiments, we preconditioned Nrf2 knockout (KO) mice and induced stroke three days later. As expected, neither sensorimotor function (Fig. 2A) nor cognitive function (Fig.

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2B-D and Supplementary Fig. 1B) was preserved by IPC in Nrf2 KO mice. The measurements of brain tissue loss at 35 d post-stroke (Fig. 2E) further confirmed the reduction of IPC-mediated

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protection in Nrf2 KO mice. Collectively, these findings indicate that Nrf2 plays an important

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role in IPC-mediated long-term ischemic tolerance in vivo.

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In vitro PC neuroprotection against OGD is associated with mild oxidative stress As mentioned in the Introduction, among the ten categories of Nrf2 activators

18

, the only

endogenous activators are lipid electrophiles, the end products of natural lipid peroxidation. Our previous work confirmed that IPC leads to the generation of sublethal amounts of 4-HNE, which then activates Nrf2 in mouse brains

17

. In the present study, we examined whether this outcome

also mediates PC-afforded neuronal protection in vitro. Consistent with previous reports, PC protected primary neuronal cultures from lethal OGD, as indicated by live/dead staining and cell counting (Fig. 3A-B), as well as the MTT and LDH release assays (Fig. 3C-D)

39

. In addition, PC also increased the levels of 4-HNE modified

proteins (Fig. 3E) and HO-1, a reliable marker of Nrf2 activation

20, 31

(Fig. 3F). Notably, the

Journal Pre-proof increase in 4-HNE and HO-1 by PC was lower than with lethal OGD (Fig. 3E-F), suggesting that PC only elicits mild or sublethal oxidative stress, consistent with our in vivo findings

17

. These

data support the hypothesis that PC elicits a sublethal degree of oxidative stress and protects against OGD-induced neuron death.

Nrf2 plays a key role in PC-mediated neuroprotection in vitro Next, we determined whether Nrf2 was critical in PC-afforded neuroprotection using

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neurons derived from Nrf2 KO fetal mice. As expected, PC-mediated protection was abolished in Nrf2 KO neurons, as evidenced by Live/Dead staining and cell counting (Fig. 4A-B), as well

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as MTT (Fig. 4C) and LDH assays (Fig. 4D), indicating an indispensable role for Nrf2 in PC.

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Pharmacological activation of Nrf2 robustly preconditions the brain in vivo A major goal in researching the mechanisms behind IPC is to translate the corresponding

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mechanism through pharmaceutical approaches

40, 41

. To determine if the Nrf2- mediated

protective mechanism is clinically translatable, we activated Nrf2 using the compound CDDO-

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TFEA, a potent triterpenoid with high blood-brain barrier permeability

42

. As shown in Fig. 5,

CDDO-TFEA pretreatment improved sensorimotor function (Fig. 5A-B) and learning and

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memory up to 35 days post- injury (Fig. 5C-D), without affecting gross motor functions

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(Supplementary Fig 1C). As expected, these neurological improvements were associated with reduced brain tissue loss (Fig. 5E). These results suggest that Nrf2 is an attractive target for pharmacological preconditioning against stroke in vivo.

Discussion The present study is the first to demonstrate that IPC provides long- lasting neuroprotection up to 35 days after stroke. IPC preserves both the functional and structural integrity of the brain in an Nrf2-dependent manner. At the cellular level, PC confers neuronal ischemic tolerance by lipid electrophile generation and Nrf2 activation. Finally, the use of CDDO-TFEA revealed that the Nrf2-mediated protective mechanism might be clinically translatable.

Journal Pre-proof Previous studies have reported that both rapid and delayed IPC only elicit short-term 4, 43

protection against global ischemia

. In focal cerebral ischemia models of embolic or

thrombotic stroke, long- lasting protection has only been reported following non- ischemic preconditioning, such as lipopolysaccharide 7 , spreading depression 8 , and asphyxia

6, 9

; thus,

whether long- lasting protection can be elicited by delayed ischemic PC was not known. Here, we show that delayed IPC confers long-term protection against stroke at both the structural and functional levels, as indicated by cognitive tests and infarct volume measurements up to 35 days after stroke.

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Our findings of spatial memory tests are particularly important because post-stroke

a heavy socioeconomic burden

11, 44

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cognitive impairment and dementia are increasingly prevalent in the elderly population and pose . The mechanisms of cognitive protection are unclear. Our

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results show that there no obvious CA1 neuronal loss, consistent with reports that the

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hippocampus is supplied by the posterior cerebral artery but not by the middle cerebral artery (MCA), and that MCAO does not cause frank hippocampal damage. However, MCAO causes 45-47

, and IPC reduces white matter injury, including the fibers that connect

the hippocampus and thalamus

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white matter damage

48, 49

. Aside from the hippocampus, other brain regions are also 50

, and the

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critical for learning and memory, such as the frontal, parietal, and temporal lobes

MCA supplies the blood to these regions. As IPC significantly reduces infarct volumes after

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MCAO, the preserved brain regions likely contribute to the improvement of cognitive function. However, further experiments are needed to elucidate the exact mechanisms underlying

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cognitive protection in IPC.

Upon exploration of the mechanism underlying IPC-afforded neuroprotection, we focused on oxidative signaling and de novo protein synthesis. We observed that several Nrf2-controlled proteins are upregulated after IPC, including HO-1, glutamate-cysteine ligase, and the thioredoxins. When Nrf2 gene expression was ablated, ischemic tolerance was lost in the present study, consistent with previous studies revealing an indispensable role for Nrf2 been reported to mediate preconditioning with hyperbaric oxygen inhaled anesthetic gas

53

51

23

. Nrf2 has also

, limb ischemia

52

, and

. Thus, the engagement of the Nrf2 pathway serves as a generalizable

mechanism underlying ischemic tolerance against stroke.

Journal Pre-proof We previously found that IPC leads to a mild increase in 4-HNE and widespread Nrf2 activation in the brain. Cortical neurons displayed a diffuse, albeit mild increase in HO-1 expression after IPC in mice

17

. In the present study, we further reported that PC protected

neurons via Nrf2 activation and that this may be mediated by the generation of lipid electrophiles. To our knowledge, we are the first to report the involvement of low levels of electrophiles in PCmediated neuronal Nrf2 activation and self-protection against ischemic injury. It is not fully understood how Nrf2 activation after IPC offers long- lasting protection against ischemic brain injury. De novo protein synthesis, controlled by transcription factors, is essential 2, 54

. A key component of IPC is the interval between

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for the emergence of ischemic tolerance

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IPC and lethal stroke, which is necessary for subsiding the sublethal injury of IPC and the synthesis of new proteins. Nrf2 is a transcription factor that upregulates anti-oxidative enzymes . Unlike small molecular antioxidants (glutathione, vitamin C, vitamin E) that are consumed

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13, 55

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during their anti-oxidative actions, anti-oxidative enzymes are not consumed during antioxidative reactions, as they operate catalytically; therefore, they exert long-term anti-oxidative

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effects 56 . In addition to these enzymes, Nrf2 also upregulates anti-apoptotic proteins and several cell-type-specific proteins, which may also contribute to the long- lasting protection of IPC.

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These pre-existing cytoprotective proteins may reduce ischemic brain injury after lethal MCAO, especially in the penumbra regions. However, it is important to note that Nrf2 may not be the

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only transcription factor that contributes to IPC-mediated protection. Peroxisome proliferatoractivated receptor-gamma (PPARγ)

57

, heat shock factor-1

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also serve to reestablish redox and proteostatic equilibrium as PPARγ, can also be activated by lipid electrophiles

60, 61

58

59

, and hypoxia- inducible factor-1

. Other transcriptions factors, such

.

There are two major goals of IPC studies. First, a superior understanding of the biological mechanisms underlying preconditioning is expected to accelerate the development of new neuroprotective strategies. To this end, pharmacological activation of Nrf2 with CDDO-TFEA for three days significantly reduced neurobehavioral dysfunction and infarct volume induced by focal ischemic stroke. These findings support our previous report that pretreatment with CDDO Im protects hippocampal CA1 neurons after global ischemia in rats

31

. Pharmaceutical

preconditioning with alternative Nrf2 activators such as sulforaphane, triterpenoid derivatives, or fish oil

31, 62

also holds translational promise. Triterpenoid derivatives or fish oil might be

especially useful in patients with a history of transient ischemic attacks or at high risk for stroke.

Journal Pre-proof Post-ischemic treatment with potent electrophiles to expedite and enhance Nrf2 activation has translational potential and may be more effective in combination with reperfusion therapy

31

.

The second goal of IPC studies is to guide its application in the clinic. Clinical investigations have shown that IPC can induce ischemic tolerance in humans

63-65

. Based on

current diagnostic approaches and techniques of ischemic stroke, however, it is difficult to predict when a stroke will occur, even in persons with obvious risk factors. Although this limits the use of IPC before the stroke onset to attenuate ischemic brain injury, t here are also several scenarios in which IPC can be purposefully induced in the clinic. For example, IPC can be

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induced in patients with anticipated ischemic events, such as vascular neurosurgery 3, 66 . Another

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tool to reduce stroke morbidity and infarct size in the clinic is remote preconditioning, which can be administered non- invasively and inexpensively with a blood pressure cuff

67

. Finally,

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pharmaceutical preconditioning and alternative electrophiles may also offer promise in the clinic,

or fish oil

20, 31, 68-70

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especially those with strong potency, such as triterpenoid derivatives, sulforaphane, resveratrol, . The latter method can be especially useful in patients with a history of

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transient ischemic attacks or at high risk for stroke. Future studies to determine Nrf2 activation in fibroblasts or plasma cells harvested from remotely preconditioned patients might also shed light

Summary

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on the underlying mechanisms.

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In conclusion, we report here that IPC- mediated ischemic tolerance provides long- lasting protection against ischemic brain injury. Based on this study and our previous work, it seems likely that IPC elicits the generation of lipid electrophiles that activate the Nrf2 pathway and induce long- lasting protection. These mechanisms are applicable to the clinic, as oral administration of CDDO-TFEA provided a similar degree of long-term protection. Therefore, targeting Nrf2 is a promising strategy to protect the mammalian brain against stroke.

Journal Pre-proof Acknowledgements Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under Award Number R01NS092810. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Jun Chen was supported by VA merit review grants and the VA Senior Research Career Scientist Award. We thank Pat Strickler

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for the administrative support.

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Conflicts of interest

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None.

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Figure legends

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Visible abstract

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(A) Ischemic stroke is associated with severe oxidative stress, which induces the generation of large amounts of lipid electrophiles and produces long-term cognitive deficits and brain tissue

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loss. (B) We discovered that ischemic preconditioning (IPC) provides enduring protection against long-term stroke- induced sensorimotor deficits, cognitive deficits, and brain tissue loss.

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In comparison with stroke, IPC only creates mild oxidative stress, which leads to the generation of subtoxic levels of lipid electrophiles followed by Nrf2 activation. This mechanism has translational potential, as the Nrf2 inducer CDDO elicits comparable protection in vivo.

Journal Pre-proof Figure 1. IPC improves long-term stroke outcomes in vivo. IPC was induced in mice by 12-min MCAO, and stroke was induced by 60- min MCAO. (A) The latency to fall off the rod was recorded up to 5 weeks post-stroke in the Rotarod test. (B-D) Morris water maze tests were performed at 31–35 d after stroke. (B) Representative traces of swim paths of mice attempting to find a submerged platform (upper panels, defined as “learning”), and searching for the platform after its removal (lower panels, defined as “memory”). (C) The escape latency was recorded on days 31–35 after stroke, and (D) the time spent in the target quadrant after the platform was removed was plotted to reflect spatial memory. (E)

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Representative MAP2 staining and analyses of brain tissue loss at 35 d after stroke. Data are

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shown as mean ± SD, n=7-9 per group. *p0.05 vs Sham, #, ## p0.05, 0.01 for IPC+Stroke vs

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Stroke.

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Figure 2. Key role of Nrf2 in IPC-mediated long-term neuroprotection in vivo. IPC was induced in Nrf2 KO mice by 12-min MCAO, and stroke was induced by 60- min

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MCAO. (A) The latency to fall off the rod was recorded up to 5 weeks post-stroke in the Rotarod test. (B-D) Morris water maze tests were performed at 31–35 d after stroke. (B) Representative

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traces of swim paths of mice attempting to find a submerged platform (upper panels, defined as “learning”), and searching for the platform after its removal (lower panels, defined as “memory”).

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(C) The escape latency was recorded on days 31–35 after stroke, and (D) the time spent in the

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target quadrant after the platform was removed was plotted to reflect spatial memory. (E) Representative MAP2 staining and analyses of brain tissue loss at 35 d after stroke in mice. Data are shown as mean ± SD, n=7-9 per group. *p0.05 vs Sham. n.s. no significant difference between Stroke and IPC+Stroke mice.

Figure 3. Preconditioning (PC) protects against OGD-induced neuronal death and is associated with moderate oxidative stress. Primary rat neuronal cultures were subjected to PC (12-min OGD) and lethal OGD (1-h OGD). (A) Representative Live/Dead cell staining, (B) dead neuron counting, (C) MTT assay, and (D) LDH release assay demonstrate that PC reduces neuronal death induced by lethal OGD. Total cell lysates were harvested for Western blots or Western slot blots. (E) Western slot blot

Journal Pre-proof showing a moderate and robust generation of 4-HNE modified proteins by PC and OGD, respectively. (F) Western blot showing a moderate and robust increase in HO-1 expression after PC and OGD, respectively. n=3-6 per group. *, **, *** p0.05, 0.01. 0.001 vs Ctrl. #, ### p0.05, 0.001 vs OGD. &&, &&& p0.01, 0.001 vs PC. Scale bar: 10 μm.

Figure 4. Key role of Nrf2 in PC-mediated neuroprotection in vitro . Primary mouse neurons from Nrf2 KO mice were subjected to PC (12-min OGD) and lethal

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OGD (1-h OGD). (A) Live/Dead cell staining, (B) dead neuron counting, (C) MTT assay, and (D) LDH release assay demonstrate that PC fails to elicit neuroprotection in Nrf2 KO neurons. n=3-6,

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*** p0.001 vs Ctrl; n.s.: not significant. Scale bar: 10 μm.

Figure 5. Pharmacological activation of the Nrf2 pathway provides long-term

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protection against cerebral ischemia in vivo.

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Mice were pretreated with CDDO-TFEA for three days and then subjected to stroke. (A) The Rotarod test and (B) adhesive removal test show that CDDO improves sensorimotor functions. (C, D) In the Morris water maze test, both learning and memory abilities are improved by CDDO.

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(E) Representative MAP2 staining and quantitative analyses of brain tissue loss at 35 d. Data are

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shown as mean ± SD, n=7-9 per group. *, **, ***p<0.05, 0.01, 0.001 vs. vehicle (Veh).

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Supplementary Figure 1. Swim speeds in Morris Water Maze tests. IPC and stroke did not alter swim speeds in (A) WT or (B) Nrf2 mice. (C) CDDO treatment also failed to change swim speeds. These results indicate that changes in learning and memory were not due to alterations in gross motor function.

Supplementary Figure 2. Hippocampal NeuN+ neurons after MCAO in mice. Immunostaining of NeuN in the ipsilateral hippocampus from MCAO and IPC+MCAO groups in WT mice. High power images of CA1 neurons are shown in the lower right inset. NeuN+ cell counting failed to reveal any difference between the two groups. Scale bar = 200 μm.

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