Propofol Ameliorates Ischemic Brain Injury by Blocking Toll-like Receptor 4-dependent Pathway and Suppressing Consequent In�ammatory Cytokine Production

Propofol suppresses tumor necrosis factor-alpha biosynthesis in lipopolysaccharide-stimulated macrophages possibly through downregulation of


Abstract
Background Ischemic stroke is one of the leading causes of mortality and morbidity worldwide. Accumulated evidence suggests that the consequent excessive in ammation plays detrimental roles in the pathogenesis of secondary injury after cerebral infarction and exacerbates the brain tissue damage. Although regulation of the in ammation would be the potential strategy for the novel treatment option, effective methods that control the cerebral in ammation have not yet been established. Recent studies have suggested that propofol, a sedative agent widely used for management of patients with acute stroke, suppresses excessive in ammation and may have neuroprotective effects against ischemic brain injury. However, the available evidence is still limited and controversial, and the underlying mechanism remains unclear. This study aimed to investigate the neuroprotective effects of propofol against ischemic brain injury, with a speci c focus on Toll-like receptor 4 (TLR4), the critical mediator of in ammation in the ischemic brain.

Conclusions
Propofol attenuates brain injury by blocking the TLR4-dependent pathway and suppressing proin ammatory cytokine production. This insight into the mechanism underlying the neuroprotective effect of propofol against ischemic brain injury may lead to a new strategy for preventing exacerbation of cerebral infarction.

Background
Ischemic stroke often results in irreversible brain tissue damage and neuronal dysfunction and has become one of the leading causes of death and long-term disability worldwide. 1 Outcomes of ischemic stroke has been improved over the past decades, and the thrombolytic therapy with tissue plasminogen activator (tPA) is currently the most e cacious therapeutic option. However, due to its narrow therapeutic windows and serious complication caused by reperfusion injury, the clinical use of tPA remains limited. 2 Although thrombolytic therapies could reduce the initial volume of brain infarction, the restored blood ow triggers in ltration of in ammatory cells to the peri-infarct area, leading to excessive local in ammation and consequent tissue injury called secondary injury. 3 Regulation of the brain in ammation would be the potential strategy for the novel treatment option. However, effective methods that control the cerebral in ammation have not yet been established. Novel pharmacological therapies that target the secondary injury is therefore desperately needed. Ischemic brain injury is also one of the most serious potential complications during medical intervention under sedation/anesthesia. Surprisingly, in recent studies using magnetic resonance imaging, the incidence of ischemic brain injury associated with noncardiac surgery was as high as 10% in patients with cardiovascular risk factors. 4 There are various causes including hypotension, hypoxia, arrhythmia, systemic in ammation, and blood loss that jeopardize cerebral circulation. In addition, because patients are unconscious during sedation/anesthesia, promptly detecting acute ischemic brain injury is challenging. Consequently, only a limited number of patients that are under sedation/anesthesia receive reperfusion therapy during the acute phase.
Propofol, a γ-aminobutyric acid agonist, provides short-acting sedative effects and has been widely used in various clinical situations, including sedation/anesthesia during medical interventions and management of patients with acute stroke. Some earlier studies using animal models of ischemic stroke have suggested the potential neuroprotective effects of propofol. 5,6 The evidence is still limited and controversial however, 7 and the mechanisms underlying the possible neuroprotective effects of propofol remain unclear. Several recent studies suggest that propofol blocks Toll-like receptor 4 (TLR4), the key mediator of in ammation, and suppresses in ammatory cytokine production. 8-10 Given that in ammation is a crucial factor in secondary injury after brain ischemia, and that TLR4 plays a pivotal role in in ammatory response in the ischemic brain, 11, 12 the TLR4-dependent pathway may be involved in the possible neuroprotective effects of propofol reported in the literature.
Thus, in this study we tested the hypothesis that treatment with propofol attenuates ischemic brain injury via inhibition of the TLR4-dependent pathway and suppression of consequent in ammatory cytokine production. The primary outcome of this study was infarct volume; the secondary outcome is proin ammatory cytokine mRNA expressions.

Animals
Male C57BL/6 mice (Japan SLC, Tokyo, Japan) and male TLR4 knockout mice on a C57BL/6 genetic background (Oriental Bio Service, Kyoto, Japan) were used in the study. All mice were aged 8-12 weeks and weighed 20-25 g, and they were group-housed at 23 °C ± 2 °C with free access to food and water and a 12-hour light/dark cycle. Ischemic brain injury Focal brain ischemia was induced via a combination of permanent left common carotid artery occlusion and distal left middle cerebral artery (MCA) coagulation. 13 Brie y, mice were anesthetized with 2-3% iso urane and placed in a dorsal position. The left common carotid artery was isolated and ligated via ventral middle neck incision. Mice were then placed in the lateral position, and a 2-mm burr-hole craniectomy was performed with a microdrill (Ideal Microdrill; Bio Research, Nagoya, Japan) between the left orbit and the left ear. The distal left MCA was exposed and coagulated using a small vessel cauterizer (Fine Science Tools, Inc., CA, USA) followed by transection of the artery. During the surgery, rectal temperature was maintained at 37 °C ± 0.5 °C with a thermostat-regulated heating pad. Brains were removed 24 hours after the induction of ischemic brain injury for real-time polymerase chain reaction (PCR) and at 7 days for measurement of infarct volume.

Propofol treatment
Propofol (10 mg/kg, 1% Diprivan, Aspen Japan, Tokyo, Japan) diluted with fat emulsion by 10 times (10 µL/g, Intralipos, Otsuka Pharmaceutical, Tokyo, Japan) was administered over 30 seconds via the tail vein 10 minutes prior to MCA occlusion. In control mice, an equal volume of the fat emulsion alone was administered in the same manner. The dose was chosen based on previously published data. 14

Measurement of Infarct Volume
Seven days after MCA occlusion and after hemodynamic measurements, mice were deeply anesthetized with 5% iso urane and euthanized via cervical dislocation. Brains were removed and cut into 1-mm-thick coronal sections. The brain slices were immersed in 2% 2,3,5-triphenyltetrazolium chloride (Sigma Aldrich, St. Louis, MO, USA) at 37 °C for 15 minutes in a dark room. The infarct area was traced and measured using image analysis software (ImageJ, National Institutes of Health, Bethesda, MD, USA). To correct for the contribution of edema, the infarct volume was calculated as follows: [1 -(total ipsilateral hemisphere -infarct region) / total contralateral hemisphere] × 100%. 15 Total infarct volume was calculated as the sum of all infarct areas multiplied by section thickness. For this assay, each group consisted of 10 mice.

Hemodynamic measurements
Heart rate and arterial blood pressure were measured non-invasively using a tail-cuff monitor (Softron, Tokyo, Japan) to evaluate the effects of propofol on hemodynamics. Values were recorded 1 hour before MCA occlusion (baseline), 10 minutes after the injection of either propofol or the control solution, and 1 hour after MCA occlusion.

Real-time PCR
Real-time PCR was used to measure mRNA expression levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor α (TNF-α). The target mRNAs from the brain slices of propofol-treated wild-type mice were compared with from those of control wild-type mice. The RNeasy Mini Kit (Qiagen, Hilden, Germany) was used for extraction of total mRNA from brain slices. One microgram of mRNA was reverse transcribed into complementary DNA with a QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). PCR was performed on a StepOne™ real-time PCR system (Life Technologies, Carlsbad, CA, USA) using the PowerSYBR® Green PCR Master Mix and corresponding primers to quantify target genes. The relative changes were expressed as a ratio to glyceraldehyde-3-phosphate dehydrogenases mRNA of the same sample. The data were analyzed using the 2 −ΔΔCT method. The 2 −ΔΔCT value of the target transcript from Page 5/15 each mouse was normalized with those of the control group mice as 1.0. The normalized 2 −ΔΔCT values derived from the two group were then compared (n = 15 each).

Statistical analysis
Statistical analysis was performed using Prism 7 software (GraphPad Software, San Diego, CA, USA). Infarct volume was assessed using two-tailed Student's t test; cytokine expression levels were assessed using Mann-Whitney U test; heart rate and blood pressure were assessed using two-way analysis of variance for repeated measures. For infarct volume and cytokine expression level, the sample size was calculated to detect signi cance with 95% con dence, assuming alpha of 0.05 and power of 0.8 (G*Power 3.1.9.3). Values are presented as mean ± standard error of mean for infarct volume, heart rate, and blood pressure, and median ± quantile for cytokine mRNA expression level. All were considered statistically signi cant for p-value of less than 0.05.

Propofol reduced cerebral infarct volume
To investigate the effects of propofol on ischemic brain injury, 8-12-week-old wild-type mice were treated with either propofol or 10% fat emulsion (control) 10 minutes before the induction of ischemic brain injury. Propofol-treated mice exhibited signi cantly smaller infarct volumes than control mice 7 days after ischemic brain injury (7.9 ± 1.4 vs. 12.6 ± 1.1 mm 3 , n = 10 each, p < 0.05; Fig. 1). Propofol treatment did not affect the hemodynamic parameters signi cantly (Table 1). There were no signi cant differences in heart rate and blood pressure among each group.
Data were presented as mean ± standard error of mean. (n = 10 each) HR, heart rate; MCAO, middle cerebral artery occlusion; TLR4KO, toll like receptor 4 knockout Propofol treatment and reduced pro-in ammatory cytokine expressions after ischemic brain injury To investigate the possible involvement of anti-in ammatory action of propofol in the neuroprotective effect observed, mRNA expression of pro-in ammatory cytokines was measured 24 hours after ischemic brain injury. As shown in Fig. 2, the propofol-treated mice exhibited lower mRNA expression levels of proin ammatory cytokines (IL-6: 0.57 ± 0.23 vs. 1.00 ± 0.39, p < 0.05, IL-1β: 0.53 ± 0.24 vs. 1.00 ± 0.36, p = 0.087, n = 15 each) compared with the control mice.

Neuroprotective effects of propofol were abolished by depletion of the TLR4-dependent pathway
In experiments testing the effects of propofol treatment against ischemic brain injury in TLR4 knockout mice, there was no signi cant difference in the infarct volume between the propofol-treated TLR4 knockout mice and control-treated TLR4 knockout mice (12.4 ± 1.6 mm 3 vs. 12.1 ± 2.0, n = 10 each, Fig. 3). Similar to the experiments using wild-type mice, there were no signi cant differences in hemodynamic parameters between two groups ( Table 2). There were no signi cant differences in heart rate and blood pressure among each group.
Data were presented as mean ± standard error of mean. (n = 10 each) HR, heart rate; MCAO, middle cerebral artery occlusion; TLR4KO, toll like receptor 4 knockout

Discussion
In the present study, treatment with propofol was associated with reduced pro-in ammatory cytokine expressions at 24 hours after ischemic brain injury, the peak timepoint of those mRNA expression levels 16 and attenuates ischemic brain injury, suggesting that propofol exerts neuroprotective effects via its antiin ammatory capacity. Because propofol at the dose used did not affect the hemodynamic parameters investigated, the effects of propofol on cardiovascular function were not likely to be involved in the neuroprotection observed in the present study.
The immune system is promptly activated once ischemic brain injury occurs. 16 Among various in ammatory mediators, TLR is considered crucial in innate immune system as a rst-line defense and mediator of in ammation. 17,18 Exogenous/endogenous TLR ligands such as heat shock proteins, brinogen, and components of the extracellular matrix are upregulated by ischemic brain insult.
Consequently, those ligands then activate TLR4, 19,20 the TLR shown to be the pivotal in ammatory mediator in the pathogenesis of ischemic brain injury. 11,12 It has been reported that while activation of TLR4 exacerbates cerebral infarction, 11 inhibition of the receptor suppresses pro-in ammatory responses and attenuates brain injury. 12 In the current study, the neuroprotective effect of propofol against ischemic brain injury was abrogated by depletion of TLR4, indicating that the TLR4-dependent pathway was substantially involved in the mechanism underlying that neuroprotective effect. The results of the current study are concordant with recent in vivo studies in which propofol suppressed in ammatory cytokine production via inhibition of TLR4-dependent pathways in various disease models, including lung, 8 liver, 9 and gastric injuries 10 and asthma. 21 Several in vitro studies using lipopolysaccharide-treated microglia, 22 macrophages, 23 spinal astrocytes, 24 and alveolar epithelial cells 25 also suggest that the anti-in ammatory action of propofol involves blocking the TLR4-dependent pathway and consequent pro-in ammatory cytokine production.
Our result showed signi cantly lower IL-6 mRNA expression levels in the brains of propofol-treated wildtype mice than in the brains of control mice, which is consistent with an earlier study in which there was a correlation between infarct volume and IL-6 mRNA expression in the brain. 26 Among various proin ammatory cytokines, IL-6 plays pivotal roles in local in ammation and cytotoxicity after ischemic brain injury, and is involved in the mechanism underlying the expansion of ischemic brain injury. 27,28 Blockade of IL-6 receptors has been shown to reduce infarct volume and improve cognitive function in an experimental model of ischemic stroke. 29 These earlier studies are concordant with the suppression of IL-6 by propofol observed in the current study after ischemic brain insult, as well as the reduction of infarct volume.
Based on the collective results of the previous studies and the current study, it is reasonable to surmise that propofol exerts neuroprotection against ischemic brain injury by blocking TLR4 and suppressing consequent production of pro-in ammatory cytokines, particularly IL-6. The clinical importance of the present study is that the results indicates that bolus propofol administration prior to ischemic insult can protect against ischemic brain injury, presumably by blocking the TLR4-dependent pathway. This understanding of the mechanism underlying the neuroprotective effect of propofol against ischemic brain injury may lead to a novel strategy to prevent exacerbation of ischemic brain injury.
With regards to study limitations, only relatively young male mice were used. Further studies should test aged mice and female mice at different menopausal stages, because those biological variables can affect the outcomes of ischemic brain injury.

Conclusions
Propofol attenuates brain injury by blocking the TRL4-dependent pathway and suppressing proin ammatory cytokine production. This understanding of the mechanism underlying the neuroprotective effect of propofol against ischemic brain injury may lead to a new strategy to prevent exacerbation of cerebral infarction.

Consent for publication
Not applicable.
Availability of data and material The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was supported by Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 20K09238). The funding bodies had no role in the design of the study, the collection, analysis, and interpretation of data, or the writing of the manuscript.
Authors' contributions KM conceived the study, acquired, analyzed, and interpreted the data, and drafted the manuscript. MK conceived the study, performed experiments and statistical analysis, and drafted the manuscript. SH, AT and SM performed experiments and helped prepare the manuscript. TI and TM performed statistical analysis and revised the manuscript for important intellectual content. All authors have read and approved the nal manuscript. Figure 1 (A) Staining for 2,3,5-triphenyltetrazolium chloride in representative 1-mm-thick coronal sections.
Arrowheads indicate the infarct area (white). (B) Infarct volumes 7 days after induction of ischemic brain injury. Mice in the propofol group mice exhibited smaller infarct volumes. *p < 0.05.

Figure 2
In ammatory cytokine expression levels 24 hours after the induction of ischemic brain injury. The 2−ΔΔCT value of the target transcript from each mouse was normalized with the median 2−ΔΔCT value from the control wild-type mice as 1.0. Mice in the propofol group exhibited lower levels of in ammatory cytokine expression. *p < 0.05.