Melatonin against acute ischaemic stroke dependently via suppressing both inflammatory and oxidative stress downstream signallings

Abstract This study tested the hypothesis that melatonin (Mel) therapy preserved the brain architectural and functional integrity against ischaemic stroke (IS) dependently through suppressing the inflammatory/oxidative stress downstream signalling pathways. Adult male B6 (n = 6 per each B6 group) and TLR4 knockout (ie TLR4−/−) (n = 6 per each TLR4−/− group) mice were categorized into sham control (SCB6), SCTLR4−/−, ISB6, ISTLR4−/−, ISB6 + Mel (i.p. daily administration) and ISTLR4−/− + Mel (i.p. daily administration). By day 28 after IS, the protein expressions of inflammatory (HMBG1/TLR2/TLR4/MAL/MyD88/RAM TRIF/TRAF6/IKK‐α/p‐NF‐κB/nuclear‐NF‐κB/nuclear‐IRF‐3&7/IL‐1β/IL‐6/TNF‐α/IFN‐γ) and oxidative stress (NOX‐1/NOX‐2/ASK1/p‐MKK4&7/p‐JNK/p‐c‐JUN) downstream pathways as well as mitochondrial‐damaged markers (cytosolic cytochrome C/cyclophilin D/SRP1/autophagy) were highest in group ISB6, lowest in groups SCB6 and SCTLR4−/−, lower in group ISTLR4−/− + Mel than in groups ISTLR4−/− and ISB6 + Mel and lower in group ISB6 + Mel than in group ISTLR4−/− (all P < .0001). The brain infarct volume, brain infarct area and the number of inflammatory cells in brain (CD14/F4‐88) and in circulation (MPO+//Ly6C+/CD11b+//Ly6G+/CD11b+) exhibited an identical pattern, whereas the neurological function displayed an opposite pattern of inflammatory protein expression among the six groups (all P < .0001). In conclusion, TLR inflammatory and oxidative stress signallings played crucial roles for brain damage and impaired neurological function after IS that were significantly reversed by Mel therapy.


| INTRODUC TI ON
Previous studies [1][2][3][4][5] have demonstrated that acute ischaemic stroke (IS) elicits a vigorous inflammatory reaction, augments the generation of cytokines and oxidative stress and initiates the complement cascade. These factors, in turn, further aggravate brain damage after acute IS, 1-7 causing irreversibly neurological sequelae. [1][2][3][4][5][6][7] Of these inflammatory mediators, damage-associated molecular patterns (DAMPs) have been identified as a critical signal that triggers the inflammatory immune system in response to tissue damage. [8][9][10] DAMPs initiate signalling cascades that activate Toll-like receptors (TLRs) and myeloid differentiation factor 88 (MyD88), and further activate the downstream signalling of IκB, nuclear factor (NF)-κB, interleukin (IL)-β, tumour necrosis factor (TNF) and interferon (IFN) regulatory factors, etc. [8][9][10][11] Some investigators have further reported that expressions of human TLR4 in plasma were associated with poor outcome and stronger inflammatory response in acute IS. 12 Melatonin (Mel) and its derivatives are characterized with potent-free radical scavengers 13,14 and have been displayed to play a key role in retaining cell membrane stability and ensuring cell survival in a toxic environment mainly through reduction in susceptibility to oxidative stress and free radical damage as well as suppression of inflammatory reaction. [15][16][17][18][19] Intriguingly, abundant experimental studies have previously shown that Mel treatment effectively reduced brain infarct volume and preserved neurological function and the integrity of brain architecture in different species of animals after acute IS. [20][21][22][23] Surprisingly, although the benefit of Mel on protecting the brain against the IS damage has been extensively investigated in animal model of acute IS, there is a lack in any clinical study to investigate whether Mel treatment would be safe and offers additional benefit for the patients after IS. This issue raises the fundamental requirement to more completely understand the mechanism for why the Mel would alleviate the brain infract volume and preserve the neurological function in setting of IS prior to conducting such a clinical trial.
Interestingly, some investigators have revealed that Mel attenuated TLR4-mediated inflammatory response through MyD88-and Tolllike receptor-associated activator of interferon (TRIF)-dependent signalling pathways in animal model of ovarian cancer 24 as well as Mel protected the myocardium against brain death tissue extract damage mainly through suppressing the DAMP-TLR2/TLR4-mediated MyD88-NF-κB inflammatory downstream signalling. 10 Accordingly, this study tested whether Mel treatment improved neurological outcome in mouse IS through inhibiting DAMP-TLR-MyD88/TRIFmediated inflammatory and oxidative stress downstream signallings.

| Animal grouping and rationale of mel dosage and time-points of treatment
Pathogen-free, 12-week-old male C57B/L6 (ie B6) mice (n = 18, ie 6 animals per each B6 subgroup) and TLR 4 10,[17][18][19]24 with minimal modification. Melatonin, which is safe and non-toxic in human beings, is popularly utilized as a dietary supplement. Previous randomized control trial had demonstrated that intravenous administration of 50 mg Mel was safe for patients with acute myocardial infarction. 25 Based on the above-mentioned information, an extrapolation to the human dosage of 5 to 10 mg Mel for daily should be very safe.

| The genetic background of TLR4 −/− mice
B10Scn-Tlr4lps-del/JthJ) used in the present study were purchased from Jackson Laboratory. Although the original genetic background of TLR4 −/− mice was C57BL/10Scn function displayed an opposite pattern of inflammatory protein expression among the six groups (all P < .0001). In conclusion, TLR inflammatory and oxidative stress signallings played crucial roles for brain damage and impaired neurological function after IS that were significantly reversed by Mel therapy.

K E Y W O R D S
inflammation, ischaemic stroke, melatonin, oxidative stress, Toll-like receptors F I G U R E 1 Pilot study for assessment of inflammatory downstream signalling in shRNA-silencing TLR4 N2a cells and shRNA-silencing TLR2/TLR4 (ie double silencing) N2a cells undergoing the BE or Mel treatment. Upper panel: Illustrating the protein expressions of MAL, MyD88, TRAF6, IκB-α and phosphorylated (p)-NF-κB, that is the inflammatory signalling, in control and shRNA-TLR4 N2a cells undergoing brain tissue extracts from ischaemic stroke B6 mouse (BE) and melatonin (Mel) treatment. A, As compared with the wild-type (WT) (ie control group of N2a cells), the protein expressions of these parameters were notably suppressed in shRNA-TLR4 N2a cells. B, On the other hand, the expressions of these parameters were notably increased in N2a cells undergoing the BE treatment that were partially reduced in shRNA-TLR4 N2a cells undergoing BE treatment. C, As compared to the control group (ie WT), the protein expressions of these parameters were remarkably suppressed by Mel treatment. However, these parameters did not show notably change in shRNA-TLR4 N2a cells undergoing the Mel treatment, implicating that Mel suppressed inflammatory signalling relying on the presence of TLR4. Lower panel: Illustrating the protein expressions of the inflammatory signalling, in control and shRNA-TLR4 N2a cells and shRNA-silencing TLR2/4 (ie TLR2 and TLR4 double knockdown) N2a cells undergoing the BE or Mel treatment. The results from (D), (E) and (F) demonstrated that as compared with the corresponding results from (A), (B) and (C), respectively, these protein expressions were further notably suppressed in shRNA-silencing TLR2/4 N2a cells and lesser change by BE or Mel treatment in shRNA double silencing of TLR2 and TLR4 condition. MAL = MyD88 adaptor-like; MyD88 = myeloid differentiation primary response 88; TRAF6 = tumour necrosis factor (TNF) receptorassociated factor 6; TLR = Toll-like receptor. IκB = nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor; NF = nuclear factor and crossed to B6 (C57BL/6), the utilization of C57BL/6 mice as the wild-type control for TLR4 −/− mice was also found in other recent study. 26,27 Hence, we suggest that the genetic background of mice should not affect the results and conclusion of the present study.

| shRNA TLR2/TLR4 double knockdown N2a cells for assessment of TLR-dependent inflammatory downstream signallings
shRNA clones were obtained from the National RNAi Core Facility

| Procedure of acute is, and advantage and disadvantage of this model
The procedure for the experimental model of acute IS has been described in our previous studies 28,29 with some modifications.
Each animal was anaesthetized by 2% inhalational isoflurane.
After exposure of the left common carotid artery (LCCA) through a transverse neck incision, a small arteriotomy was performed on the LCCA through which silicon rubber-coated monofilament (ie 0.22 ± 0.01 mm diameter) was carefully advanced into the distal left internal carotid artery for occlusion of the left middle cerebral artery, causing brain ischaemia and infarction of its supplied area.
The nylon monofilament was removed 50 minutes after occlusion, followed by closure of the muscle and skin in layers. For groups 1/2 (sham-operated control), only neck skin and muscle layers were opened, followed by closing these two layers. The advantage of this animal model is reproducible and could provide consistently neurological dysfunction for the study. On the other hand, the

| Corner test for assessment of neurological function prior to and after is induction
The sensorimotor functional test (corner test) was conducted for each rat of each group (ie n = 6 per group) at baseline and on days 1, 3, 7, 14 and 28 after acute IS induction as we previously described. 28,29 In detail, the mice could walk through a tunnel and then turn into a 60-degree corner. To exit the corner, the mice could turn either left or right. The results were recorded by a technician blinded to the study design. This test was repeated 10 to 15 times with at least 30 seconds between each trial. We recorded the number of right and left turns from 10 successful trials for each animal and used the results for statistical analysis.

| Procedure and protocol of brain magnetic resonance imaging (MRI) for determining the brain infarct volume (BIV)
The procedure and protocol for brain magnetic resonance imaging (MRI) study were based on our previous report. 29 The MRI was performed at day 28 after IS induction. Briefly, during MRI measurements, mice were anaesthetized by 2% inhalational isoflurane with room air and placed in an MRI-compatible holder (Biospec 94/20). Rectal temperature and respiration were monitored throughout the procedure to ensure normal physiological conditions were maintained.

| Immunofluorescent (IF) staining
The procedure for IF staining has been described in our previous reports. 29

| Western blot analysis
The procedure for Western blot analysis was based on our recent reports. 28 The washing procedure was repeated eight times within one hour.
Immunoreactive bands were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences) and exposed to BioMax L film (Kodak). For the purpose of quantification, ECL signals were digitized using Labwork software (UVP).

| Statistical analysis
Statistical analysis was adequately performed by ANOVA followed by Bonferroni multiple comparison post hoc test. SAS statistical software for Windows version 8.2 (SAS institute) was utilized. A probability value <.05 is considered statistically significant.

| Na2 cell culture with IS B6-EX to mimic the preclinical setting of brain-damaged released DAMPs for interacting with TLR2-and TLR4-mediated inflammatory downstream signalling pathways that suppressed by Mel (Figures 2 and 3)
Based on the pilot study results of Figure 1, we utilized the cell cul-

| Utilized the preclinical study to further elucidate the impacts of DAMP-TLR2/TLR4 interaction and Mel on regulating the upstream and downstream signalling pathways (Figures 4-7)
To assess the principal roles of DAMP-TLR4 and Mel on regulating  Figures 8 and 9, we summarized the proposed underlying mechanism of oxidative stress downstream signalling pathway participated in brain damage in setting of acute IS. Mel = melatonin; ROS = reactive oxygen species (ROS); ASK1 = apoptosis signal-regulating kinase 1; MMK = mitogen-activated protein kinase kinase; DRP1 = dynamin-related protein 1

F I G U R E 11
Brian MRI determined the brain infarct volume by day 28 after acute IS induction. A-F, Illustrating the brain magnetic resonance imaging (MRI) finding for identification of brain infarct zone of being greater in group 4 than in group 5, but these parameters showed no difference between groups 1 and 2 (Figures 4 and 5).
Interestingly, when the subgroup analysis was examined, we found that these parameters were remarkably reduced (ie about 50.0%) in group 4 (IS TLR4−/− mouse ), further remarkably reduced (about 60.0%) in group 5 (IS B6 mouse + Mel) and furthermore remarkably reduced (>85.0%) in group 6 (IS TLR4−/−mouse + Mel) (Figures 4 and 5), suggesting that a net further reduction of these biomarkers in the same species (ie TLR4 −/−mouse ) was about 35.0% in group 6 as compared to group 4. Additionally, this finding (ie a net further reduction) may implicate that Mel treatment reduced the expressions of these parameters through not only TLR4 but also TLR2 and possible other signallings (although much less important) such as other TLRs.
Another interesting finding in the present study was that the protein expressions of these parameters did not differ between groups 4 (ie IS TLR4−/−mouse ) and 5 (IS B6 mouse + Mel), implying that the of p-NF-κB (ie located in cytosol) and nuclear-NF-κB (ie located in nucleus), two downstream signalling conductors of IKB-γ/α/β, exhibited an identical pattern of MyD88 among the six groups ( Figure 5).
The protein expressions of p-IRF3/p-IRF7 (ie located in cytosol) and nuclear-IRF3/nuclear-IRF7 (ie located in nuclei), four indices of terminal inflammatory signalling conductors, exhibited an identical pattern of MyD88 among the six groups ( Figure 6).
Finally, the protein expressions of TNF-α, IL-1β, IL-6 and IFN-γ, four translation of cytokines (ie end-products) by nuclei/DNA generation in response to the inflammatory signalling stimulations, exhibited an identical pattern of MyD88 among the six groups ( Figure 6).
Accordingly, based on the findings from Figures 1-6, we summarized the proposed underlying mechanism of DAMP-TLR inflammatory downstream signalling pathway of acute IS that was suppressed by Mel treatment (Figure 7).

| Protein expressions of oxidative stress signalling pathway, mitochondrial-damaged biomarkers and cell death factors by day 28 after is induction (Figures 8 and 9)
The protein expressions of NOX-1 and NOX-2, two indicators of oxidative stress, were lowest in groups 1 and 2, highest in group 3, significantly higher in groups 4 and 5 than in group 6, but they showed no significant difference between groups 1 and 2 or between groups Accordingly, based on the findings from Figures 8 and 9, we summarized the proposed underlying mechanism of oxidative stress downstream signalling pathway involved in brain damage in setting of acute IS ( Figure 10). Figure  11)

| BIV by day 28 after acute is induction (
The brain MRI demonstrated that the BIV was lowest in groups 1 and 2, highest in group 3, significantly higher in group 4 than in groups 5 and 6 and significantly higher in group 5 than group 6 but it showed no significant difference between groups 1 and 2 ( Figure 11).

| Time courses of neurological function examinations and the BIA (Figure 12)
By day 14 after IS induction, the TTC stain demonstrated that the BIA was lowest in groups 1 and 2, highest in group 3, significantly higher in group 4 than in groups 5 and 6 and significantly higher in group 5 than in group 6 ( Figure 12).
By day 0, the neurological function (ie by corner test) did not differ among the six groups. However, by day 3, the neurological function was significantly impaired in groups 3 to 6 than in groups 1 and 3, but it showed no different among the former four groups or between the latter two groups. Furthermore, by days 7, 14 and 28, the neurological function was significantly impaired in group 3 than in groups 1 and 2, while significantly progressively improved in group 5 and further improved in groups 4 and 6.

| The 3rd day's circulating levels of inflammatory cells and inflammatory cell infiltrations in BIA by day 28 after acute is induction (Figure 13)
By day 3 after acute IS procedure, the circulating numbers of myeloperoxidase (MPO)+, Ly6C+/CD11b+ and Ly6G+/CD11b+ cells, three indices of inflammation, were lowest in groups 1 and 2, highest in group 3, significantly higher in group 4 than in groups 5 and 6 and significantly higher in group 5 than in group 6. Additionally, by day 28 after IS procedure, the cellular expressions of CD14 and F4/80 in BIA, another two indicators of inflammation, displayed an identical pattern to the circulating inflammatory cells among the six groups ( Figure 13). The most novel finding in the present study was that the brain MRI (ie imaging study) TTC stain (ie pathological finding) demonstrated that the BIV was substantially reduced in groups 4/5 and more substantially reduced in group 6 as compared with group 2.

| D ISCUSS I ON
These findings could explain why the neurological outcome was much better in groups 4 to 6 than in that of group 3.
It was noteworthy that as compared with group 3 animals, a great reduction in both BIA and BIV was found in group 4 animals, highlighting that TLR4 played a critical role on participating the brain organ damage in setting of IS. Another noteworthy point was that as compared with group 4, wide margins of reduction in BIA and BIV were found in group 5, highlighting that Mel was not inferior to the role of deleting TRL4 on protecting brain integrity against ISinduced damage. Of most distinctive issue was that the BIA and BIV were much lower in group 6 than in group 5 (ie ratio of brain infarct volume reduction in IS TLR4−/−mouse + Mel vs IS B6 mouse + Mel >1.0fold) implicating that not only TLR4 (despite its role was proved to be dominant by the current study) but also TLR2 (ie proved by our previous study) 10 as well as other possible contributors combined to involve in the destruction of brain architecture and deterioration of neurological function after acute IS.
The associations between generation of oxidative stress and mitochondrial damage and cell apoptosis and organ damage in setting of ischaemia have been extensively addressed by previous studies. 6,7,10,15,[17][18][19]28 An essential finding in the present study was that the oxidative and mitochondrial-damaged markers were notably increased in IS animals. Additionally, the oxidative stress downstream signalling pathway (refer to Figure 5) was clearly delineated in these IS animals (ie group 3). Importantly, all of these parameters were notably reduced in groups 4/5 and further notably reduced in group 6 animals. Accordingly, our findings, being consistent with the findings of previous studies, 6,7,10,15,[17][18][19]28 not only could explain why the brain damage was notably lesser and the neurological function was more preserved in groups 4 to 6 animals but also could explain our hypothesis that "other contributors gathered together" to involve in the brain destruction and neurological dysfunction after acute IS.
It is well recognized that inflammation aggravates brain damage after acute IS, 1-7 causing irreversibly neurological sequelae. [1][2][3][4][5][6][7] A fundamental finding in the present study was that the cellular-molecular levels (ie in tissue and circulation) of inflammatory mediators were remarkably increased in group 3 animals as compared with groups 1/2, highlighting that these terminal signallings of inflammatory mediators were the results of DAMP-TLR interaction after acute IS. Our findings, in addition to reinforcing the findings of previous studies, 1-7 could, at least in part, explain why the brain damage was remarkably present in these IS animals. Conformingly, the cellular-molecular perturbations and brain damage were substantially reduced, whereas the neurological function was notably preserved in groups 4/5 and further improved in the group 6.

| S TUDY LIMITATI ON
This study has limitations. First, the study period was 28 days.
Accordingly, the long-term effect of Mel therapy on protecting brain from IS damage is currently unclear. Additionally, the pathophysiological changes were completed at 28 day after ischaemia.
Theoretically, short-term studies (acute phase) are more valuable for signalling analyses, especially when innate inflammatory immune-related downstream signalling was taken into investigations. However, this study did not provide this information. Second, the limitation for the clinical translation of the treatment with melatonin was that the Mel treatment began 3 days prior to the IS. Third, the behavioural experiments did not investigate in the present study that would, therefore, affect the precise and comprehensive estimation of neurological recovery after Mel treatment.
In conclusion, Mel therapy effectively preserved the integrities of brain architecture and neurological function in setting of acute IS in mice mainly through regulating the inflammatory and oxidative stress signalling pathways.

ACK N OWLED G EM ENTS
This study was supported by a program grant from Chang Gung Memorial Hospital, Chang Gung University (Grant Number: CMRPG8H0061). We especially gratefully thank Dr Ching-Hua Hsieh to provide the TLR 4−/− mice as the gift to us for the study.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest associated with this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets of present study can be available from the corresponding author upon request (han.gung@msa.hinet.net).