Introduction
Cerebral ischemia (CI) is a pathological condition characterized by rapid loss of blood flow to the brain often due to stroke or cardiac arrest. In selectively vulnerable brain regions, such as the hippocampus, CI typically leads to irreversible cellular injury/death as a consequence of impaired ion homeostasis, massive cell depolarization (anoxic depolarization, AD), and excitotoxicity [
1]. In the USA alone, approximately 795,000 new/recurrent strokes and more than 356,000 out-of-hospital cardiac arrests are reported each year [
2]. Notably, surviving patients often exhibit debilitating cognitive impairments, spanning across multiple domains including attention, memory, language, perceptual motor, and executive functioning [
3‐
5]. Unfortunately, there are currently no treatment interventions available to facilitate cognitive recovery after injury.
While the central mechanisms underlying post-CI cognitive impairments have not been fully elucidated, evidence indicates that synaptic dysfunction plays a major role. It has been well established that synaptic compartments undergo early structural and functional alterations following ischemia-induced excitotoxicity. In fact, synaptic failure—manifested as spine loss, aberrant spine morphology, and impaired synaptic plasticity—is evident despite the presence of viable neurons after ischemia [
6‐
10]. Early and persistent deficits in synaptic function have been attributed to several mechanisms [
11‐
15], including pathological effects mediated by the actin-binding protein, cofilin [
16]. Cofilin, an important cytoskeletal protein involved in modulating actin dynamics, is regulated via its phosphorylation status at serine residue 3 (Ser 3), whereby dephosphorylation promotes its activation. Under conditions of oxidative stress, cofilin hyperactivation and subsequent binding with actin induce the formation of stable cofilin-actin bundles, referred to as “rods,” which have been shown to disrupt normal actin dynamics and synaptic structure, induce synapse loss, block axonal and dendritic transport, and exacerbate mitochondrial membrane potential loss [
17‐
19]. Unsurprisingly, previous studies have demonstrated that exposure to CI induces cofilin hyperactivation and cofilin-actin rod formation [
16,
20‐
22]. To this end, there exists a critical need for the development of therapies targeting early disturbances in synaptic function and their underlying cause after CI.
The utilization of prophylactic strategies, which serve to benefit a large subset of individuals with high proclivity to CI, offers a promising means to combat rapid disturbances in synaptic function given their ability to mitigate deleterious mechanisms of injury at the earliest time possible. Our lab has previously demonstrated that pharmacological preconditioning with the compound resveratrol (3,5,4′-trihydroxystilbene; RSV), herein referred to as resveratrol preconditioning (RPC), promotes ischemic tolerance and renders the brain resistant to subsequent, lethal ischemic insults [
23,
24]. RSV is a naturally occurring phytoalexin commonly found in several dietary foods, which has garnered considerable interest as a therapeutic agent against synaptic dysfunction over the years. Across several neurological conditions, studies have demonstrated improvements in hippocampal long-term potentiation (LTP), spine density, and learning/memory following RSV treatment [
25‐
28]. Notably, RSV has been shown to modulate the expression of important synaptic-related proteins, including the activity-regulated cytoskeleton-associated protein (Arc) [
29,
30]. Although well known for facilitating activity-dependent endocytosis of AMPARs [
31,
32], Arc has also been shown to influence the phosphorylation status of cofilin during activity-dependent states [
33]. Previous studies in our laboratory have shown that Arc is required for the protective effects mediated by other forms of pharmacological preconditioning [
34]; however, a role for Arc in RPC-induced neuroprotection has yet to be defined. Moreover, RPC-mediated effects on overall synaptic function have not been previously explored in the context of ischemic injury.
In the present study, we aimed to investigate the effects of RPC on early ischemia-induced excitotoxic processes and synaptic dysfunction in the mouse hippocampus. Utilizing ex vivo brain slices, which serve as a suitable model system to study early electrophysiological changes during/after ischemia, we first examined changes in AD onset latency, intracellular calcium accumulation, synaptic transmission, and synaptic plasticity following injury. Additionally, we sought to elucidate potential mechanisms underlying RPC-mediated effects on synaptic function. Given Arc’s protective role in other preconditioning paradigms and its regulatory effect on phospho-cofilin levels, we hypothesized that RPC may protect against synaptic damage during ischemia by upregulating Arc expression and mitigating cofilin hyperactivation. Taken together, our study provides novel evidence supporting the use of RPC as a neuroprotective strategy to combat early CI-induced synaptic dysfunction.
Materials and Methods
Animals
All animal usage and experimentation were approved by the Institutional Animal Care and Use Committee at the University of Miami and were in accordance with the US Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals and the National Research Council’s Guide for the Care and Use of Laboratory Animals. Euthanasia methods were consistent with the American Veterinary Medical Association (AVMA) guidelines. Male wild-type C57BL/6 J mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) between 7 and 8 weeks of age. Animals were housed in an AAALAC-accredited facility and maintained on a 12/12-h light/dark cycle at constant temperature and humidity. Mice were given free access to food and water ad libitum. Upon receipt from Jackson Labs, animals were allowed to acclimate for at least 1 week prior to experimental manipulations and numbered for identification by ear punch. Only mice between the ages of 8 and 12 weeks were used for experiments.
Drug Preparation and Treatments
Trans-resveratrol (Sigma-Aldrich, St Louis, MO, USA) was prepared as previously described [
29]. Briefly, resveratrol was dissolved in 100% DMSO at a concentration of 65 mg/mL and aliquots were stored at – 20°C in amber tubes to minimize light exposure. Immediately before use, stock solutions were diluted to a 1 mg/mL working solution (1.5% DMSO) with saline (0.9% NaCl). A single intraperitoneal injection (i.p.) of 10 mg/kg of resveratrol or vehicle (DMSO) was administered to animals approximately 48 h prior to experimental manipulations.
Acute hippocampal slice preparation
Mice were anesthetized by inhalation of a gas mixture of isoflurane in 30% oxygen (300 mL/min) and 70% nitrous oxide (700 mL/min) delivered by a vaporizer. Mice were then euthanized and their brains were quickly removed with the entire head submerged into ice-cold artificial cerebral spinal fluid (ACSF) bubbled with carbogen (95% O2/5% CO2). The ASCF solution was prepared by mixing the following components (in mM) in nanopure water: 4.5 KCl, 2 MgSO4 • 7H2O, 1.25 Na2HPO4 • 7H2O, 126 NaCl, 2 CaCl2, 26 NaHCO3, 10 glucose (all chemicals from Millipore-Sigma). The ACSF solution was saturated with carbogen and, if necessary, the pH was adjusted to 7.40–7.45 (305–312 mOsm) with HCl. The dissected brain was then placed into an ice-cold slurry of carbogenated ACSF solution and allowed to sit for 1 min. Sagittal slices of 300 μm thickness were sectioned using a Leica VT1000S microtome (Leica Microsystems, Nussloch, Germany) and then transferred into a submerged-type holding chamber containing cold ACSF gassed with carbogen. Slices were gradually brought up to room temperature and allowed to incubate for at least 1.5 h prior to use for experiments. Of note, slices prepared for determination of cytosolic calcium levels were sectioned in sucrose cutting solution containing the following (in mM): 3 KCl, 7 MgCl2, 1.25 Na2HPO4, 60 NaCl, 0.5 CaCl2, 28 NaHCO3, 5 glucose, 110 sucrose). After sectioning, the hippocampus was dissected out from the slice and placed into a holding chamber containing a 50:50 mixture of sucrose-based ACSF and standard ACSF (composition in mM: 2.5 KCl, 1 MgCl2, 1.25 Na2HPO4, 125 NaCl, 2 CaCl2, 25 NaHCO3, 10 glucose) for 20 min. Thereafter, slices were transferred to a separate holding chamber containing standard ACSF and allowed to recover for 1.5 h.
Following the preincubation period, acute slices were transferred into an interface-type recording chamber (Harvard Apparatus, Boston, MA) perfused with carbogenated ACSF (flow rate: 1.5 mL/min) and allowed to acclimate for 20 min prior to recordings. The temperature was maintained at 34°C using an automated temperature controller (Warner Instruments, Holliston, MA) and all relevant equipment were positioned on a vibration isolation table (Technical Manufacturing Co., Peabody, MA) with a surrounding Faraday cage to prevent electrical and mechanical noise. Schaffer collaterals were electrically stimulated with a bipolar tungsten electrode (TST53A05KT; Word Precision Instruments, Sarasota, FL, USA) and stimulus pulses (0.1 ms duration) were generated using a S48 square pulse stimulator equipped with a SIU5 Stimulus Isolation Unit (GRASS Technologies). Evoked field excitatory postsynaptic potentials (fEPSPs) were measured in the stratum radiatum of the CA1 hippocampal subfield with glass microelectrodes filled with 150 mM NaCl (2.5–5 MΩ). Microelectrodes were pulled from borosilicate glass capillaries (1B150-4; World Precision Instruments) with a Sutter P-87 Micropipette Puller (Sutter Instruments, Navato, CA, USA). Signals were amplified using an Axopatch 200B amplifier (Molecular Devices, San Jose, CA, USA)—low-pass filtered at 10 kHz—and digitized at a sampling frequency of 10 or 20 kHz using a Digidata 1200 series interface (Molecular Devices) coupled with Clampex 9 software (PClamp, Molecular Devices). Acquired data were analyzed offline using Clampfit 10.7 software (PClamp, Molecular Devices).
At the beginning of each experiment, input/output (I/O) relationships were determined for each slice. I/O curves were generated by gradually increasing the stimulus strength until the maximal evoked response was reached. The stimulus intensity was adjusted to evoke a fEPSP of about 35–40% of the maximum slope. The negative-going slope of the fEPSP—measured over the 20–80% range between the start of the fEPSP and fEPSP peak amplitude—was used as an index of synaptic strength. In all recordings, a presynaptic fiber volley (FV) preceded the fEPSP; thus, we considered the first point immediately after the fiber volley as the start of the fEPSP. Synaptic transmission was assessed by measuring the relationship between the FV amplitude and fEPSP slope over increasing stimulus intensities obtained from the I/O protocol. The fEPSP slope values were plotted against presynaptic FV amplitudes for each slice. Each set of plotted data was then fit to a linear regression to determine the I/O mean slope for each slice, which was then averaged per group.
To determine changes in paired-pulse facilitation (PPF), a pair of stimulus pulses were delivered to slices over several intra-pulse durations—25, 50, 100, and 150 ms. The paired-pulse ratio (PPR) was measured by dividing the slope value of the fEPSP elicited by the second pulse (S2) by the slope value of the fEPSP elicited by the first pulse (S1). A PPR (S2/S1) value greater than 1 indicated the occurrence of PPF. To assess LTP, baseline fEPSP responses were recorded for 10–30 min, after which LTP was induced using a theta-burst stimulation (TBS) protocol (three trains of stimuli delivered 15 s apart and each train consisting of 10 high-frequency bursts (100 Hz) delivered at 5 Hz) and evoked potentials were recorded (1 stimulus every 30 s) for 50–60 min. Slices that exhibited ≥ 20% baseline variance were excluded from further analysis. Post-TBS values are expressed as the fold change of the average fEPSP slope obtained from baseline recordings. LTP data were analyzed from an average of 11 traces at three selected time intervals (first, middle, and last 5 min of the recording after delivery of TBS). A maximum of two slices were used per animal and each slice was considered an n = 1.
Oxygen and Glucose Deprivation and Anoxic Depolarization
Ischemia was induced ex vivo via oxygen and glucose deprivation (OGD), in which oxygenated ACSF containing glucose was replaced with glucose-free ACSF gassed with 95% N
2/5% CO
2. For electrophysiological studies, O
2 was also replaced with N
2 in the gaseous phase of the interface-type recording chamber. Acute slices were subjected to OGD until the onset of AD, which was reflected by a large negative direct current (DC) shift in the extracellular field potential. Immediately after AD onset, the medium was switched back to normal oxygenated ACSF; thus, the duration of AD, referring to the time between AD onset and reinstatement of oxygen and glucose, was kept constant at 0 min. We opted to terminate OGD at the onset of AD, rather than use a fixed OGD duration, in order to control for the extent of damage endured by each slice. As the period of time in which OGD persists beyond the onset of AD determines whether synaptic responses recover and cellular injury becomes irreversible [
35‐
37], controlling for the duration of AD offers a better means to maintain similar ischemia-induced changes across slices. After OGD, slices were allowed to recover for 1 h in which evoked fEPSPs were recorded at a rate of 1/30 s.
For both calcium and protein assessments, acute slices were transferred to a homemade submerged-type chamber in which slices rested on cell culture inserts (Millipore-Sigma) or custom-made nylon-mesh inserts. The chamber was placed inside a miniature incubator (Bioscience Tools, Highland, CA, USA) and maintained at 34°C. For induction of ischemia, slices were transferred to wells containing glucose-free ACSF gassed with 95% N2/5% CO2 for varying durations. In Sham conditions, slices were transferred to a separate chamber containing normal carbogenated ACSF. For protein expression studies, acute slices were harvested immediately following OGD and the hippocampus was dissected out for lysate preparation.
Intracellular Calcium Measurements
Relative changes in intracellular calcium concentrations were measured spectrophotometrically using a leakage-resistant form of the calcium sensitive fluorescent ratiometric dye, fura-2 AM, known as fura-PE3 AM (Millipore-Sigma). Since fura-PE3 retains nearly identical spectral properties as fura-2 [
38], relative calcium concentrations were determined by the ratio of the emission intensity (510 nm) excited by 340 nm and 380 nm measured using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA USA). For bulk loading of the dye in acute slices, we adapted a protocol from a previously published study [
39]. Fura-PE3 AM was freshly prepared for each experiment, in which 50 µg of the dye was dissolved in 9 µL DMSO and 1 µL Pluronic F-127 (20% solution in DMSO; ThermoFisher Scientific, Rockford, IL, USA) and vortexed thoroughly for at least 10 min to make a 4 mM solution. The stock solution was directly pipetted onto each slice in carbogenated Ca
2+-free ACSF solution. Application of fura-PE3 in this manner resulted in an initial high concentration of fura-PE3 AM and final concentration of 16 µM in the entire chamber. Slices were loaded in the dark for 50 min at 37°C and washed for 45 min in a separate holding chamber. Following the washout period, a baseline reading was taken. To induce OGD, slices were transferred to a separate submerged chamber and placed onto a custom mesh support. A final reading was taken immediately after exposure to OGD for several durations (5, 10, 15, 20, and 25 min). For a given experiment, 12–14 hippocampal slices were obtained from one animal, which was sufficient to assess cytosolic calcium changes for all OGD durations tested (Sham, 5, 10, 15, 20, and 25 min OGD) in parallel. Two slices from a single animal were pooled together for each OGD duration and was considered an
N = 1.
Subcellular Fractionation
Cellular fractions were separated using a protein subcellular fractionation kit (ThermoFisher Scientific) in accordance with the manufacturer’s instructions. Briefly, tissue was gently washed with ice-cold 1 × PBS and homogenized in a pre-chilled glass Dounce with CEB buffer containing protease and phosphatase inhibitors (ThermoFisher Scientific). The homogenate was transferred into a Pierce tissue strainer and centrifuged at 500 × g for 5 min (4°C). The supernatant was collected (cytosolic fraction) and the pellet was resuspended in ice-cold MEB buffer containing protease and phosphatase inhibitors. The resuspended pellet was vortexed vigorously for 5 s, incubated for 10 min at 4°C with gentle mixing, and centrifuged at 3000 × g for 5 min. The supernatant containing membrane proteins was collected. Pellets were rinsed twice with the appropriate buffer in between steps. Protein concentration was determined using the BioRad DC™ Protein Assay kit (BioRad, Hercules, CA) and 20–50 μg of protein was used for western blot analysis.
Whole Cell Lysate Preparation
Harvested hippocampal slices were pooled together (4–5 slices), rinsed in ACSF, and homogenized in RIPA buffer containing protease and phosphatase inhibitors (ThermoScientific) with a motor pestle (20 pulses) in an Eppendorf tube. Homogenized samples were incubated for 30 min at 4°C on a rotator and then sonicated twice at low intensity for 8 s. Samples were then centrifuged at 16,000 × g for 15 min at 4°C. The supernatant was collected and 30 µg of protein was used for western blot analysis.
Western Blotting
After protein determination, samples were mixed with 4X Laemmli sample buffer (BioRad, Hercules, CA, USA) containing β-mercaptoethanol and denatured by heating at 95°C for 5–10 min. Western blotting was performed using standard procedures as described in the Supplementary Methods.
RNA Extraction and qRT-PCR
Total RNA was extracted and purified using the Trizol/RNeasy hybrid or traditional Trizol extraction method. Detailed methods are provided in the Supplementary Methods.
Antisense Oligodeoxynucleotides
Knockdown of Arc protein expression ex vivo was achieved using antisense oligodeoxynucleotides (AS ODNs). AS ODNs and scrambled control (SCR Ctrl) ODNs were designed as detailed in previous studies [
31,
40]. The Arc AS ODN was targeted to a 20-mer sequence of the
Arc mRNA spanning the translation start site. SCR Ctrl ODNs consisted of randomized nucleotides with the same base composition as the antisense sequence. ODNs were conjugated to cholesterol triethylene glycol (CholTEG) to facilitate cellular uptake (incorporated at the 3′ end of the ODN) and contained phosphorothioate linkages between the three bases on both the 5′ and 3′ ends to confer increased resistance to degradation by endogenous nucleases. Arc AS ODN sequence (asterisks indicate a phosphorothioate bond): 5′-G*T*C*CAGCTCCATCTGGT*C*G*T-CholTEG-3′. SCR Ctrl ODN sequence: 5′-C*G*T*GCACCTCTCGCAGG*T*T*T-CholTEG-3′. ODNs were synthesized by Integrated DNA Technologies (IDT, Coralville, Iowa, IA, USA) and reconstituted in 1X IDTE solution (pH: 8.0; IDT). Upon use, ODNs were diluted in standard ACSF. To validate the efficiency of ODN uptake into cells, an identical Arc AS ODN was designed with the addition of a fluorescein (FAM) tag at the 5′ end.
Following an initial acclimation period at 34°C, acute slices were incubated with either FAM/CholTEG- or CholTEG-conjugated ODNs in a submerged-type chamber for 2 or 6 h at 35°C, respectively. A diluted stock of the AS ODN was directly pipetted onto each slice to give an initial concentration of 250 µM and a final concentration of 5 µM. Slices were then washed in ACSF for 15 min and either fixed for imaging (see slice resectioning below), harvested for protein determinations, or subjected to OGD (see above).
Slice Resectioning for Fluorescent Imaging
Following incubation with FAM/CholTEG-conjugated ODNs, acute brain slices were fixed in 4% formaldehyde (Pierce, ThermoFisher Scientific, Waltham, MA) in 1 × PBS for 1 h at 4°C on a rocker. Post-fixed slices were washed 3 × 10 min in ice-cold 1 × PBS and then incubated in increasing concentrations of sucrose in 1 × PBS (10%, 20%, and 25%) over the course of 3 days. Slices were flash frozen in liquid nitrogen-cooled isopentane (Sigma-Aldrich) in OCT medium and cut to a thickness of 14 µm using a Leica CM 1850 cryostat (Leica Biosystems, Nussloch, Germany). Coverslips were mounted with Prolong Diamond antifade mountant with DAPI (Invitrogen). Samples were imaged using an inverted Leica Stellaris confocal microscope with × 10 (N.A. 0.40) and × 20 (N.A. 0.75) objectives. Maximum intensity projections were generated from z-stacks (1024 × 1024 pixel resolution, 0.68 µm step size) taken at × 10 and × 40 magnifications (× 1 and × 2 zoom settings, respectively).
Statistical Analysis
GraphPad Prism 9.4.1 and R-4.1.1 software were used to analyze data. Assumptions of normality were examined using quantile–quantile (Q-Q) plots and the Shapiro–Wilk test. Assumptions of homogeneity of variance were assessed using Levene’s test and the Brown-Forsythe test. For simple comparisons of two sample means, an unpaired two-tailed t-test with Welch’s correction was used. For comparisons of more than two sample means, a one-way analysis of variance (ANOVA) with Dunnett’s (comparing each group to control) or Bonferroni’s multiple comparisons test was used as appropriate. Data with 2 factors were analyzed by two-way ANOVA, followed by Tukey’s HSD or Bonferroni’s post-hoc test as appropriate. LTP data from OGD studies were analyzed by a mixed-model two-way repeated measures (RM) ANOVA (between subject factor: treatment; within-subject factor: time) followed by Bonferroni’s multiple comparisons test. Correlation analyses were assessed with Pearson correlation coefficient. For electrophysiological studies, the experimenter was blinded to the treatment group during the experimental procedure and data collection. Groups were only revealed at the final analysis. Sample sizes are reported under each figure legend, in which “n” represents the number of slices per group and “N” represents the number of animals per group. All data, except for correlation analyses, are expressed as mean ± SEM. For all statistical analyses, the p criterion was 0.05.
Discussion
Early and persistent disturbances in synaptic function are thought to contribute largely to cognitive impairments following CI. Thus, the development of therapies that effectively target rapid changes in synaptic integrity and function after ischemic injury is of vital importance. The present study investigated the neuroprotective effects of a prophylactic therapy, known as RPC, against CI-induced synaptic impairments in an ex vivo slice model. We report that RPC protects against ischemia-induced excitotoxicity and synaptic dysfunction in the hippocampus as evidenced by RPC-mediated reductions in cytosolic calcium accumulation, increases in the latency to AD, resistance to hyperexcitability, and improvements in LTP shortly after induction of ischemia. In addition, we demonstrate that RPC is capable of attenuating immediate ischemia-induced hyperactivation of cofilin. This effect was partially dependent on Arc expression and offers a potential mechanism by which RPC protects against synaptic dysfunction. These findings suggest that RPC can prevent early and detrimental effects on synapses provoked by ischemia, which have implications for improving cognitive outcomes following injury.
A characteristic feature of both focal and global ischemia is the anoxic depolarization of neuronal and glial cells, which has been associated with steep rises in extracellular glutamate and intracellular calcium concentrations [
64,
65]. AD is a type of spreading depolarization that is thought to be a major determinant of irreversible injury as early studies have shown a negative correlation between the duration of AD and the recovery of the orthodromic population spike in field recordings, but no correlation between the total duration of anoxia and the recovery of synaptic responses [
37,
47,
66,
67]. Accordingly, our study was designed to isolate electrophysiological changes in response to the AD event, in which the AD duration was kept constant. While previous studies have investigated synaptic plasticity deficits following fixed periods of ischemia [
15,
54], this is the first study, to the best of our knowledge, to evaluate alterations in synaptic function and plasticity shortly after exposure to ischemia and AD onset.
Notably, AD is considered a target for therapeutic intervention, as blocking/delaying AD during ischemia markedly improves cellular recovery [
68,
69]. In the current study, we report that RPC was able to extend the latency to AD (Fig.
2A, B), suggesting a role for RPC in modulating processes related to AD generation and ensuing excitotoxicity. This has important implications in the clinical setting, as drugs that block/delay AD also have the potential to inhibit milder AD-like events, known as peri-infarct depolarizations (PIDs), that develop following stroke and contribute to a secondary injury process in the penumbra [
70,
71]. Considering that AD occurs minutes following stroke onset within the core, AD itself is not a clinical target for improving outcomes. Rather, recurrent PIDs, which occur over prolonged periods and promote infarct expansion [
70,
72], represent a feasible target for therapeutic intervention as there exists a window of opportunity to suppress their activity. Since PIDs are generated in a similar manner as AD, treatments that delay/block AD will also affect PID generation—perhaps even more effectively given that energy stores are not completed depleted in the penumbra, at least initially. Importantly, delaying PIDs likely reduces the number of PIDs triggered in a given period of ischemia; thus, maintaining energy reserves for longer durations and expanding the time window for treatments.
Our lab has previously shown that increased Arc expression induced by preconditioning with the PKCɛ activator, ψɛ-receptor of activated C kinase (ψɛRACK), is necessary for neuroprotection against ischemia and the PKCɛ-mediated delay to AD [
34]. This effect was attributed to the internalization of AMPAR GluR2 subunits via Arc, which lead to a shift in AMPAR-mediated currents. In our current study, we observe RPC-mediated upregulation of Arc protein expression particularly within the cell membrane. It is possible that similar effects on AMPAR subunits induced by ψɛRACK-mediated upregulation of Arc expression occur with RPC. However, this is unlikely as we did not find that RPC altered the expression of either AMPAR GluR1 or GluR2 subunits in the hippocampus (data not shown). Alternatively, previous studies have demonstrated that increasing glycolytic energy production or preventing depletion of intracellular ATP via creatine supplementation during ischemia significantly delays AD [
37,
73,
74]. Interestingly, our lab has previously shown an enhancement in bioenergetic efficiency following RPC during the long-term extended window of ischemic tolerance, in which basal ATP levels and mitochondrial abundance were increased [
29]. As the onset of AD reflects the time needed for ATP concentrations to fall below a certain threshold to maintain ionic pumps [
75], higher basal levels of ATP induced by RPC likely contribute to the observed delay to AD.
Effects on basal ATP levels and mitochondria may also explain RPC-mediated effects on calcium regulation during ischemia. RSV is known to activate the histone deacetylase, sirtuin1 (Sirt1), which is required for RPC-mediated neuroprotection against CI [
76]. Interestingly, Sirt1 can interact with a crucial mediator of mitochondrial biogenesis known as peroxisome proliferator-activated receptor coactivator-1α (PGC-1α) [
77]. Although not directly tested in this study, we speculate that a critical mechanism by which RPC attenuates cytosolic calcium accumulation is by promoting mitochondrial biogenesis via Sirt1 and PGC-1α. Increased mitochondrial abundance would allow for more distributed and enhanced sequestration/buffering of intracellular calcium levels, which would not only result in reduced cytosolic calcium accumulation but also a lower intramitochondrial calcium load. However, it is unlikely that RPC targets a single player involved in Ca
2+ regulation; rather, we suspect that its effects are pleiotropic and may involve multiple mechanisms resulting in decreased Ca
2+ influx, increased Ca
2+ efflux, or both. Interestingly, studies have revealed both direct and indirect RSV-mediated effects on intracellular calcium signaling mechanisms via regulation of voltage-gated calcium channels and calcium ATPases [
41]. For example, RSV has been previously shown to upregulate the expression of the sarcoplasmic calcium ATPase, which functions to maintain low cytosolic Ca
2+ levels by pumping free Ca
2+ ions into the lumen of the endoplasmic reticulum [
78]. Certainly, future studies are warranted to elucidate specific RPC targets as well as to identify the specific cell types that benefit from RPC-mediated reductions in cytosolic calcium during ischemia.
Notably, as the initial sites in which apoptotic-like events develop due to calcium overload [
79], synapses endure the earliest consequences of ischemia-induced excitotoxic injury. This includes alterations in synaptic structure, neurotransmission, and synaptic plasticity [
6,
15,
54]. Evidently, the amelioration of excitotoxic processes during ischemia has implications for preserving synaptic function/plasticity after CI. In our current study, we found that increasing durations of OGD heightened basal levels of synaptic transmission—an effect that was not present in slices derived from RPC-treated mice (Fig.
3F). While increases in synaptic transmission typically reflect enhanced synaptic efficacy under physiological conditions, excessive excitatory synaptic activation during disease states is thought to be pathological [
80,
81]. Earlier studies have reported increases in synaptic efficacy between the CA3-CA1 synapse 5–10 h following induction of CI in vivo, which were later followed by a loss of electrophysiological responses that coincided with pyramidal cell degeneration [
82,
83]. Thus, we suspect that enhancements in excitatory synaptic transmission observed acutely following OGD may exacerbate excitotoxicity after reperfusion, thereby incurring additional damage to synapses and contributing to the development of delayed neuronal death. The prevention of aberrant ischemia-induced synaptic hyperexcitability may provide a means by which RPC protects against these deleterious effects. Alterations in synaptic transmission could arise from several mechanisms, including effects on pre/postsynaptic sites or reductions in synaptic inhibition. To elucidate whether this effect was mediated by an augmented postsynaptic response, we investigated potential changes in AMPAR subunit composition within the hippocampal CA1 region (Supplementary Fig.
S3) as previous reports observed the “switching” of AMPAR subunits at the cell surface after ischemia [
13,
14,
48]. However, as we did not detect any changes in AMPAR subunit composition 1 h post-OGD, we suspect that other mechanisms may be at play. Future studies are required to identify potential targets.
In line with previous studies, we also found significant impairments in a well-established form of long-term synaptic plasticity, known as LTP (Fig.
4A–C). LTP is associated with the strengthening of synapses that leads to long-lasting changes in synaptic efficacy and is thought to underlie specific forms of associative learning and memory. Accordingly, deficits in LTP induced by disease conditions have been attributed to memory loss and cognitive decline [
84]. Although several studies have reported LTP deficits in different models of CI [
6,
7,
49‐
53], few studies have investigated very early effects of ischemia on hippocampal LTP [
15,
54]. Assessing early changes in synaptic plasticity may provide better insight into the efficacy of therapies seeking to ameliorate synaptic dysfunction and degeneration after ischemic injury. Remarkably, we found that RPC was able to rescue ischemia-induced impairments in LTP induction and maintenance, further demonstrating a protective role against synaptic dysfunction. Given that levels of basal synaptic transmission were elevated in vehicle-derived slices, we speculated whether this effect could interfere with the processes underlying LTP induction. Earlier studies have identified a pathological form of synaptic plasticity induced by ischemia known as ischemic LTP (iLTP) [
46], which is characterized by an increase in synaptic efficacy and has been previously shown to occlude physiological LTP in a rodent model of cardiac arrest [
85]. While our study did not specifically investigate this phenomenon, the observed enhancements in synaptic transmission after OGD suggest a possible role for iLTP in occluding physiological LTP. However, this does not entirely explain our findings as OGD durations varied and the majority of slices within the vehicle group did not express iLTP behavior after reperfusion, yet still exhibited severe LTP deficits.
Interestingly, we find that ischemia altered the expression of important synaptic-related proteins necessary for maintaining normal synaptic function (Fig.
6 and Supplementary Fig.
S7). Specifically, we demonstrated early ischemia-induced decreases in the expression of PSD-95, neuroligin 1, and the NMDAR2B subunit, which agree with observations reported in previous studies [
60,
86,
87]. However, as RPC failed to rescue OGD-induced reductions in their expression, we sought to investigate OGD effects on other synaptic-related proteins. As mentioned previously, RPC significantly increased the expression of Arc, which is known to interact with a host of different effector proteins in order to bidirectionally regulate synaptic plasticity. In addition to its interactions with the endocytic machinery to regulate AMPAR endocytosis, Arc has been shown to control spine morphology and structural plasticity via regulation of actin dynamics [
32]. Various mechanisms of action have been elucidated, but one interesting finding is that Arc can maintain the actin-binding protein, cofilin, in its inactive (phosphorylated) state during LTP consolidation [
33]. Cofilin is a critical regulator of actin dynamics/reorganization and under physiological conditions drives actin assembly or disassembly depending on the concentration of cofilin relative to actin. However, in highly oxidative environments, abnormally high levels of active cofilin may promote the bundling of cofilin‐actin filaments into stable rod-like structures, which form due to the generation of intermolecular disulfide bonds between cofilin molecules [
17]. Cofilin-actin rods have been detected in the brains of Alzheimer’s disease patients [
88] and hyperactivation of cofilin has been shown to contribute to LTP deficits in Alzheimer’s disease models [
89,
90]. Ischemic/anoxic insults also induce rapid hyperactivation of cofilin and subsequent cofilin-actin rod formation, which have been attributed to synaptic dysfunction after injury [
16,
20‐
22]. Likewise, we observed robust dephosphorylation of cofilin immediately after OGD exposure (Fig.
6A, B), indicating its early hyperactivation and suggesting a possible role for cofilin in mediating CI-induced impairments in synaptic plasticity.
Consistent with our hypothesis, RPC ameliorated OGD-induced cofilin dephosphorylation (hyperactivation), which was partially dependent on Arc as incubation with Arc AS ODNs blocked RPC-mediated maintenance of phospho-cofilin levels at longer OGD exposures (Figs.
6F, G and
7). Previous studies have demonstrated that Arc does not coimmunoprecipitate with cofilin, indicating an indirect regulatory effect on cofilin activity [
91]. Arc, however, does interact with drebrin A, a major regulator of cytoskeletal dynamics, which competes with cofilin for binding to actin filaments [
91]. It is possible that Arc cooperates with drebrin A to modulate cofilin activity indirectly. Alternatively, Arc may interact with signaling pathways that serve to activate/inactivate cofilin. Generally, the phosphorylation of cofilin at Ser 3 is regulated by multiple kinases and phosphatases. In adult neurons, Lim domain kinase 1 (LIMK1) primarily phosphorylates/inactivates cofilin, whereas phosphatase slingshot-1L (SSH1L) and chronophin (CIN) dephosphorylate/activate cofilin as well as LIMK1 [
17]. Previous studies have demonstrated that ATP depletion enhances CIN-dependent cofilin dephosphorylation [
92] and that ischemia-induced cofilin dephosphorylation is mediated by calcium influx and subsequent calcineurin-dependent activation of SSH1L [
20]. Arc could possibly carry out its effects by suppressing phosphatase-activating mechanisms during ischemia; however, future studies are necessary to elucidate any specific interactions with signaling cascades involved in phospho-cofilin regulation.
Notably, as knockdown of Arc was insufficient to block RPC-mediated attenuation of cofilin hyperactivation in slices subjected to 5 min of OGD, we suspect that RPC may target various signaling pathways that regulate cofilin phosphorylation independent of Arc. Given the observed reductions in cytosolic calcium accumulation during ischemia (Fig.
2E), RPC may simply repress calcineurin-dependent activation of SSH1L during the initial stages of injury. Generation of reactive oxygen species (ROS) during ischemia may also contribute to cofilin activation as previous studies have demonstrated ROS-mediated activation of SSH1L and cofilin dephosphorylation [
93]. Preconditioning or pretreatment with RSV has previously been shown to upregulate cellular antioxidants, including NAD(P)H-quinone oxidoreductase 1, methionine sulfoxide reductases A, and manganese superoxide dismutase [
23,
94,
95]. As such, RPC may limit cofilin hyperactivation by protecting against oxidative stress. On the other hand, studies have shown that Sirt1 can suppress microRNA-134, which becomes increased shortly after ischemia [
96] and has been shown to inhibit
Limk1 translation [
97,
98]. Thus, RPC could potentially sustain phospho-cofilin levels by modulating LIMK1 expression. It is likely that RPC integrates both kinase-activating and phosphatase-inactivating mechanisms during acute exposure to ischemia. During prolonged states of energy deprivation, RPC may rely on secondary defense mechanisms involving Arc to prevent cofilin hyperactivation and subsequent cofilin rod formation that may contribute to synaptic dysfunction.
While our findings highlight a novel protective role for RPC against CI and reveal possible molecular targets for intervention, our study is not without limitations. First, we acknowledge that our experiments were only performed on male mice and, therefore, do not consider potential sex differences. The initial design of this study excluded females in order to improve feasibility and simplify the experimental paradigm. As different stages of the estrous cycle have been shown to influence outcomes after CI, with protective effects observed during the estrus/proestrus stage when estrogen levels are high [
99,
100], female studies require examining RPC-mediated effects at distinct phases of the estrous cycle. This becomes further complicated when also considering the effects of estrogen levels on synaptic plasticity [
101,
102]. In light of the promising findings outlined in this work, we plan to conduct similar studies in females at variable stages of the estrous cycle in the future. Second, while changes in Arc expression and cofilin activation offer potential mechanisms by which RPC exerts its protective effects, we do not directly show that upregulation of Arc or attenuation of cofilin hyperactivation accounts for the observed preservation of synaptic function mediated by RPC. Studies incorporating knockdown of Arc in vivo following RPC are required to delineate the role of Arc against ischemia-induced synaptic dysfunction. Additionally, as we only measured p-cofilin levels immediately after ischemia, future studies examining the kinetics of p-cofilin recovery following reperfusion are warranted in order to establish a stronger link between cofilin hyperactivation and synaptic plasticity deficits, which were observed 1 h post reperfusion. Given that reperfusion itself is associated with increased ROS production [
103] and the formation of cofilin-actin rods persists in the presence of oxidative stress [
104], we expect that cofilin will remain hyperactivated for a prolonged period following ischemia/reperfusion injury. Future studies will seek to evaluate RPC’s effects on cofilin hyperactivation and rod accumulation at later time points post-injury and establish whether RPC-mediated preservation of synaptic function/plasticity requires regulation of cofilin phosphorylation status.
Despite these limitations, the work presented here lends further support to the use of RPC as a viable therapeutic strategy to limit ischemic damage and potentially improve cognitive outcomes after injury. Although it is impossible to predict the occurrence of cerebral ischemic events, RPC can be applied to a variety of clinical scenarios. For example, patients undergoing certain procedures that carry risk of causing CI, such as coronary artery bypass grafting and carotid endarterectomy, could directly benefit from RPC. In addition, our findings hold promise for the potential implementation of chronic preconditioning interventions in high-risk patients, such as those with a history of stroke/transient ischemic attacks or those with defined standard risk factors (i.e., age, genetic disposition, hypertension, diabetes). In such cases, there may be a future where pharmacological IPC mimetics, such as RPC, can be taken for prolonged periods in a similar manner as antiplatelet treatments, which are currently being used for long-term secondary stroke prevention [
105]. Notably, the maximal window for neuroprotection following the administration of a single dose of resveratrol in mice is 14 days [
106]. We have not tested RPC beyond this interval; however, it is possible that the neuroprotective effects mediated by RPC can persist for longer periods—potentially sustained by a positive-feedback mechanism. Certainly, more research in the translational application of preconditioning is warranted to confirm its effectiveness in humans as well as a more defined therapeutic window that is relevant for individuals with high proclivity to CI.