Stroke has a multifactorial etiology, and narrow treatment time windows have restrained the efficacy and safety of existing therapeutic applications because effective therapy can only be achieved within 4 to 6 hours after stroke.1 Inflammation plays a critical role in stroke.2 Cerebral ischemia-reperfusion injury induced by the infiltration of microglia/macrophages, neutrophils, and lymphocytes in ischemic brain tissues is related to the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway.2–4 Furthermore, PI3K/Akt pathway activation enhances cell survival and promotes neuronal survival after cerebral ischemia.5–8 Furthermore, high-mobility group box-1 (HMGB1) is a key proinflammatory cytokine and neuroimmune system regulator, which is involved in the pathogenesis of cerebral ischemic injury.9,10
Emulsified isoflurane (EI) is a novel emulsified preparation of isoflurane, which can be administered IV without a specific vaporizer.11,12 EI has been investigated in the first in-human volunteer phase I clinical trial, which aimed to evaluate its safety, tolerability, and anesthetic efficacy.13 EI can produce cardiac, kidney, liver, and lung protection against ischemia-reperfusion injury.14–18 Furthermore, isoflurane inhalation has been demonstrated to decrease cerebral ischemia through the PI3K/Akt pathway.5,6,19 EI comprises IV administered isoflurane, which can induce protection of multiple organs. Thus, we hypothesized that pretreatment with EI protects against focal ischemic brain injury.
In the current study, we sought to determine whether IV EI pretreatment protects against focal ischemic brain injury by a middle cerebral artery occlusion (MCAO) model in rats and whether its protective effects are associated with the PI3K-Akt signaling pathway via attenuation of HMGB1 release after stroke.
The study protocols were approved by the Medical Faculty Ethics Committee of Zhejiang University. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats (300–350 g), 12 to 14 weeks old, were provided ad libitum water and food before the experiment.
Focal Cerebral Ischemia and Neurologic Evaluation
The experimental procedure is summarized in Figure 1A. Rats were anesthetized with chloral hydrate and allowed to adapt for 15 minutes to attain a steady state for baseline and spontaneously breathe oxygen-enriched air (fraction of inspired oxygen: 40%) through a facemask. EI or vehicle was infused IV for 30 minutes. The rectal temperature was maintained at 37 ± 0.5°C with a heating pad. Transient focal ischemia was induced by MCAO for 100 minutes.20 In brief, we introduced a silicone-coated 4-0 monofilament (Doccol Corp, Redlands, CA) into the left external carotid artery and advanced it from the carotid bifurcation to occlude the middle cerebral artery for 100 minutes; the filament was then removed. Sham-operated rats underwent an identical procedure, with the exception of monofilament insertion.
The neurologic score was measured 48 hours after MCAO according to a neurologic grading scale previously described21: 0, no observable neurologic deficit (normal); 1, failure to extend left forepaw upon lifting the whole body by the tail (mild); 2, circling to contralateral side with normal posture at rest (moderate); 3, leaning to contralateral side at rest (severe); and 4, no spontaneous motor activity. The evaluator was blinded to the treatment.
Forty-eight hours after MCAO and immediately after neurologic scoring, rats were euthanized with carbon dioxide and decapitated.22 Brains were removed and sectioned into coronal slices. The sections were incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC). The infarct volume (percentage of hemispheric volume) was determined by a blinded observer and corrected for edema by using ImageJ software (version 1.61; National Institutes of Health, Bethesda, MD) as previously described.23,24
Intralipid® (30%), provided by Huarui Pharmacy, Ltd. (Chengdu, China), was used to dissolve liquid isoflurane (Abbott Laboratories, Queenborough, Kent, United Kingdom) and served as the vehicle for the test suspension preparation. EI was provided by Yichang Humanwell Pharmaceutical Co., Ltd. (Hubei, People’s Republic of China) and prepared by dissolving liquid isoflurane in 30% Intralipid at a 1:11.5 volume ratio with an 8% isoflurane concentration (vol/vol).25
In the first experiment, rats received an isovolumetric IV infusion of 8 mL/kg/h of Intralipid as the vehicle or 4 different doses of EI (1, 2, 4, or 8 mL/kg/h) via the caudal vein for 30 minutes before MCAO.
In the second experiment, rats were injected with IV Intralipid or EI at 4 mL/kg/h for 30 minutes based on the previous results. To determine whether Akt inhibition abolishes EI protection, 10 μL of the PI3K inhibitor LY294002 (10 mM) or vehicle was infused into the ventricular space ipsilateral to the ischemia (from bregma: anteroposterior, −0.92 mm; mediolateral, 1.5 mm; dorsoventral, 3.5 mm) before treatment.26 The infarct size was measured 48 hours after stroke via TTC staining.
Rats were euthanized with carbon dioxide and perfused with ice-cold phosphate-buffered saline 48 hours after MCAO, followed by 4% paraformaldehyde.21 The brains were removed, postfixed for 48 hours in 4% paraformaldehyde, and cut into 50-μm coronal sections. The sections were incubated at 4°C with anti-HMGB1 antibody (diluted 1:200; ab18256; Abcam, Cambridge, MA) and anti-NeuN antibody27,28 (diluted 1:200; MAB377, Millipore, MA) or mouse anti-CD68 antibody.29,30 The sections were then rinsed and incubated for 2 hours with Alexa 594–conjugated goat anti-rabbit (for HMGB1; Invitrogen, Carlsbad, CA) or Alexa 488–conjugated goat anti-mouse (for CD68 or NeuN; Invitrogen) antibodies. The sections were mounted on glass slides using Vectashield mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Negative control experiments without primary antibodies were performed in parallel and did not exhibit staining. CD68 and NeuN expression was assessed with a Zeiss Axiovert inverted epifluorescence microscope (Zeiss LSM510, Germany). Three sections were randomly chosen per animal, and CD68+ or NeuN+ cells in predefined areas31 (Fig. 2) were counted using ImageJ. An average for each animal was subsequently calculated.
Rats were euthanized 48 hours after reperfusion. Cortical brain tissues from the boundary zone adjacent to the ischemic core (IC) in the EI and vehicle groups were dissected on ice, and proteins were extracted. Western blotting was performed as described in our previous studies.23 In each lane, 50 μg protein was loaded and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis by using a 4% to 15% Ready Gel for 1.5 hours. The protein bands were transferred. After blocking with 5% nonfat dry milk, the blot was incubated with rabbit polyclonal antibodies against phosphorylated Akt (p-Akt) (Ser473) and Akt (1:1000; Cell Signaling Technology) overnight at 4°C, followed by incubation with horseradish peroxidase–conjugated secondary anti-rabbit antibody (1:2000; Cell Signaling Technology). The immunoreactive bands were subsequently visualized with enhanced chemiluminescence, and the p-Akt and Akt bands were detected. The membranes were washed and incubated with anti-β-actin antibody (1:5000; Sigma Aldrich) to control for protein loading. The band optical densities were analyzed and normalized to β-actin using ImageJ.
Serum Collection and HMGB1 Enzyme-Linked Immunosorbent Assay
Forty-eight hours after stroke, blood without anticoagulant was collected via cardiac puncture through the right atrium, placed in a 1.5-mL conical tube, and maintained at room temperature for 30 minutes to clot. The tubes were centrifuged at 5000g for 10 minutes. The serum samples were analyzed to determine the HMGB1 content using an enzyme-linked immunosorbent assay kit (# F0102; Westang, Shanghai, China).
The sample sizes of animals were based on our primary experiment and published articles.a 23 SPSS 16.0 for Windows (SPSS, Inc., Chicago, IL) was used for statistical analysis, and the data were presented as mean ± SD. After normal distribution and equal variances among groups were tested by Shapiro-Wilk test and the residuals of the analysis of variance model were analyzed with P value >0.05 for each model, 1-way analysis of variance was used to compare differences among groups, followed by Bonferroni or Tamhane T2 post hoc test as suitable. A P value <0.05 was considered statistically significant.
Effects of EI on the Infarct Size and Neurologic Scores After MCAO
MCAO models were established, and the infarct size and neurologic scores were measured 48 hours after stroke. One milliliter per kilogram per hour (35.9 ± 9.17, n = 10, P = 0.796) and 2 mL/kg/h (28.58 ± 12.63, n = 12, P = 0.572) of EI did not affect the infarct size or neurologic scores compared with the vehicle (37.09 ± 10.46, n = 11). However, as demonstrated by the representative TTC-stained coronal sections (Fig. 1B), 4 mL/kg/h (21.08 ± 11.24, n = 12, P = 0.006) and 8 mL/kg/h (20.00 ± 6.78, n = 9, P = 0.007) of EI significantly reduced the infarct size compared with the vehicle (Fig. 1B). The neurologic scores were also significantly ameliorated via EI infusion pre-MCAO at the same doses compared with the vehicle (1.13 ± 0.48, 1.11 ± 0.55 vs 1.95 ± 0.65, P = 0.015 and 0.025, respectively) (Fig. 1C). These results demonstrated that 4 and 8 mL/kg/h of EI protected against ischemic brain injury to the same degree in this model.
Effects of EI on Neuronal Death, Inflammation, and HMGB1 Release in the Ischemic Area After Stroke
To further determine the effects of EI on neuronal death and the brain inflammation cellular response, we performed double immunostaining of CD68 (activated macrophage marker) or NeuN (marker for neuronal nuclei) and HMGB1. The results demonstrated that HMGB1 was robustly expressed in the sham group brains and was restrictively located in the nuclei. However, at 48 hours after a stroke, limited NeuN-positive cells were identified in the IC (Fig. 2B), which indicates that most neurons died after stroke, and HMGB1 was released from necrotic neurons. Accordingly, increased HMGB1 expression was identified in the serum following stroke (Fig. 3). However, CD68 and HMGB1 double-positive macrophage/microglial cells were simultaneously increased in the ischemic area, which suggests that macrophages/microglia accumulated in the IC and inflammatory cellular response in ischemic brains 48 hours after stroke (Fig. 2C). In addition, EI administration (4 mL/kg/h) before stroke significantly improved neuron survival (982.7 ± 364.4 vs 439.8 ± 278.4, n = 4 and 6, respectively, P = 0.036) and inhibited CD68+ macrophage/macroglial infiltration in the IC compared with the vehicle group (188.2 ± 49.1 vs 282 ± 49.4, n = 4 and 6, respectively, P = 0.018) (Fig. 2D). These findings suggest that EI strongly suppressed brain inflammation after stroke.
Effects of EI on the PI3K/Akt Signaling Pathway in MCAO Rats
To provide direct evidence regarding the contribution of Akt activity to the protective effect of EI pretreatment, the p-Akt and total Akt protein levels in the boundary zone adjacent to the IC were measured via Western blot. The effects of inhibiting the activity of PI3K, an upstream activator of Akt, with the specific inhibitor LY294002 were assessed based on infarct size and behavioral testing after ischemia. Western blots indicated that the p-Akt level in the EI pretreatment group (4 mL/kg/h) was increased at 48 hours compared with the vehicle group (50.33 ± 4.73 vs 37.5 ± 3.11, n = 3 and 4, respectively, P = 0.007), and this increase was inhibited by LY294002 (43.00 ± 4.84 vs 37.5 ± 3.11, n = 5 and 4, respectively, P = 0.091) (Fig. 4). Furthermore, LY294002 abolished the protective effects of EI in rats with transient focal ischemia injury, the neurologic score changes after stroke were consistent with the infarct sizes (Fig. 5).
Intracerebroventricular Injection of LY294002 on the Effects of EI on HMGB1 Release in Serum 48 Hours Poststroke
On the basis of our findings that HMGB1 was released in ischemic brains, we investigated the effect of EI on HMGB1 in peripheral blood. Enzyme-linked immunosorbent assay was used to measure the serum HMGB1 levels 48 hours poststroke. The serum HMGB1 concentration was significantly increased in the MCAO rats and was nearly 3-fold higher than the sham-operated rats 48 hours poststroke. Four milliliters per kilogram per hour EI pretreatment significantly reduced the HMGB1 serum levels compared with the vehicle group (3.62 ± 0.72 vs 5.73 ± 0.65, n = 9 and 12, respectively, P < 0.001), and LY294002 (5.31 ± 0.72 vs 5.73 ± 0.65, n = 9 per group, P = 0.216) abolished the effects of EI (Fig. 3).
These findings demonstrated that EI reduced the infarct size in an in vivo rat model when administered before brain ischemia. These findings suggest that EI pretreatment protected against stroke and improved spontaneous activity at 4 mL/kg/h, which activated the PI3K/Akt signaling pathway in transient focal ischemia injury. This protective effect was abolished by LY294002, a PI3K inhibitor, which adversely attenuated the inhibitory effects on serum HMGB1 release 48 hours poststroke.
In 1961, methoxyflurane emulsion was reported to induce general anesthesia in animals and humans. However, the injective volatile anesthetic was primarily criticized for its associated complications, such as thrombophlebitis in humans.32 More recently, Eger and MacLeod33 reported that EI induced rapid-onset general anesthesia without significant side effects. Consistent with this finding, our recent first in-human volunteer phase I clinical trial demonstrated that IV EI is safe in humans within the range of 0.3 to 64.6 mL/kg.13 EI produced a rapid onset of unconsciousness in all volunteers at ≥22.6 mL/kg, followed by fast, predictable, and complete recovery.13 EI has been demonstrated to exhibit multiple organ-protective effects across systems, such as the cardiovascular and respiratory systems, after ischemia-reperfusion injury. These effects may be related to the decrease in mitochondrial DNA segment deletion and apoptosis of the myocardium, improvement of the mitochondrial antioxidant ability, increased antioxidation ability of myocardial cells as a result of increased nitric oxide synthesis, increased superoxide dismutase activity, and decreased malondialdehyde concentrations.11,14–18 Chiari et al.11 have demonstrated that myocardial protection against infarction was successfully induced by IV EI, enflurane, or sevoflurane in rabbits. In our study, EI pretreatment protected against brain injury induced by stroke. Brain injury related to 100 minutes of ischemia was significantly attenuated, and all animals survived using an EI infusion rate of 4 mL/kg/h. However, the neuroprotective effect of EI did not further increase when the dose was increased to 8 mL/kg/h. Thus, our studies contribute novel knowledge to the increasing evidence regarding the efficacy of EI as an organ-protective agent.34
Regarding cerebrovascular vasodilation of volatile anesthetics, we speculate that EI neuroprotection is partially related to increased brain perfusion. Chi et al.19 have demonstrated that isoflurane pretreatment improved regional cerebral blood flow and increased regional oxygen supply and consumption in the focal ischemic area without affecting capillary permeability during the early stage of focal cerebral ischemia.
In our study, the mechanism of the protective role of EI may have been a result of PI3K/Akt signaling pathway activation. A previous study demonstrated that isoflurane postconditioning induced protection in neuronal cultures, which was attenuated by the Akt inhibitor LY294002. These findings suggest that the postconditioning effects of isoflurane may be mediated by Akt and reduced mitochondrial membrane permeability.5,6 Our in vivo model indicated that Akt kinase activity was improved by EI, whereas LY294002 inhibited this effect. Together, EI suppresses activation and infiltration of microglia/macrophages in the IC, which suggests that EI exerts neuroprotective effects via PI3K/Akt signaling pathway activation and thereby reduces ischemia-induced brain inflammation.
HMGB1 (i.e., amphoterin or HMG-1) is a DNA-binding protein with 216 amino acids (29 kDa).35 HMGB1 has a highly conserved structure in several species. In general, HMGB1 is expressed in various vertebrate tissues, including the brain and may serve as a major upstream paracrine inflammatory mediator within the neurovascular unit in ischemic stroke.9 HMGB1 can be passively released by necrotic cells. In addition, activated macrophages can actively secrete HMGB1 for inflammatory stimulation after stroke.36 HMGB1, in turn, hastens brain inflammation, which thereby advances cerebral injury.37 Anti-HMGB1 mAb inhibited brain edema development and exhibited efficient clearance of circulating HMGB1.10 The current rat MCAO model indicated that HMGB1 was highly expressed in the ipsilateral cortex and located in the nuclei restrictively in the sham group. However, limited positive neuronal cells were identified in the IC area 48 hours after ischemia (Fig. 2B). We suggest that most neurons died after stroke, and HMGB1 was released from necrotic neurons. Furthermore, serum HMGB1 overexpression was also identified after stroke, which was significantly decreased by EI pretreatment, but attenuated by the PI3K/Akt inhibitor LY294002. Collectively, the neuroprotective actions of EI are related to the PI3K/Akt signaling pathway.
The current study has several limitations. All animals were not intubated and ventilated. EI slightly depresses respiration in rats during infusion. However, the half-life of EI is short,12 and there was a 15-minute interval that comprised the washing time for EI before MCAO. The differences in the arterial blood gas and acid-base equilibrium between groups were not significant during MCAO in the preliminary experiments. Thus, this limitation may not have affected the study results. From a translational perspective, it would not be practical to administer treatment before stroke. Nevertheless, these findings provide critical insights into the mechanisms of injury. Moreover, we only assessed the p-Akt (Ser473) protein levels and total amount of Akt. Additional investigations related to Akt subtypes are necessary to advance our understanding of the cerebral protection of EI pretreatment.
In conclusion, EI pretreatment reduces infarct size and improves neurologic function outcomes after stroke in rats. This protection is likely a result of PI3K/Akt signaling pathway activation and HMGB1 targeting, which is potentially a novel method for stroke treatment.
Name: Hongfei Zhang, MD, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and prepare the manuscript.
Attestation: Hongfei Zhang has seen the original study data, reviewed the analyzed data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Xiaoxing Xiong, MD, PhD.
Contribution: This author helped design the study, conduct the study, analyze the data, and prepare the manuscript.
Attestation: Xiaoxing Xiong has seen the original study data, reviewed the analyzed data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Jin Liu, MD.
Contribution: This author helped design the study, analyze the data, and prepare the manuscript.
Attestation: Jin Liu has seen the original study data, reviewed the analyzed data, and approved the final manuscript.
Name: Lijuan Gu, MD.
Contribution: This author helped conduct the study and collect the data.
Attestation: Lijuan Gu has seen the original study data, reviewed the analyzed data, and approved the final manuscript.
Name: Fengxian Li, MD.
Contribution: This author helped conduct the study, collect the data, and prepare the manuscript.
Attestation: Fengxian Li has seen the original study data, reviewed the analyzed data, and approved the final manuscript.
Name: Yi Wan, MD.
Contribution: This author helped analyze the data and prepare the manuscript.
Attestation: Yi Wan has seen the original study data, reviewed the analyzed data, and approved the final manuscript.
Name: Shiyuan Xu, MD.
Contribution: This author helped design the study, analyze the data, and prepare the manuscript.
Attestation: Shiyuan Xu has seen the original study data, reviewed the analyzed data, and approved the final manuscript.
This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.
a Sample size estimation was calculated based on our primary experiments and recently published articles. Taking the experiments in Figure 1 as example, in a 1-way analysis of variance study, a total sample of 40 subjects with 5 groups’ comparison achieved 90% power to detect differences among the means versus the alternative of equal means using an F test with a 0.05 significance level. The size of the variation in the means was represented by their SD, which is 6.78, and the common SD within a group was assumed to be 10.00. Furthermore, we also used a laser Doppler prone to monitor the relative regional cerebral blood flow to make sure the right place of suture and the middle cerebral artery occlusion model.
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