Once delayed non-invasive remote ischemic preconditioning protects against early stroke by modulating neuroinflammatory responses in rats

Once delayed non-invasive remote ischemic preconditioning (RIPC) has been proven to provide endogenous protection against injury induced by ischemia–reperfusion in the central nervous system. However, for thus ischemic preconditioning method, it is still unclear how long this protection can maintain and what the underlying mechanism is. In this study, we tested the hypothesis that once delayed non-invasive RIPC protects brain injury at short reperfusion time. The rat was stimulated by transient middle cerebral artery occlusion (MCAo) for 90 min, and subsequent reperfusion was performed at 6 h, 72 h and 7 days after MCAo. RIPC was conducted in both hind limbs 24 h before MCAo for 3 cycles (10 min ischemia/ 10 min reperfusion). The infarct size was measured by 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC) staining and Cresyl violet (CV) staining. The mRNA and protein levels of inflammatory cytokines in the brain were measured by real-time RT-PCR and ELISA. The results showed that once delayed non-invasive RIPC reduced the infarct size, improved neurological functions and behavioral performance at 6 and 72 h post-stroke. There was no change by reperfusion at 7 d after MCAo. RIPC reduced the levels of TNFα, IL-1β and IL-6 in the brain at 72 h post stroke. It also reduced the levels of TNFα and IL-1β when reperfusion at 6 h after MCAo. Our results strongly supported that once delayed non-invasive RIPC protects against stroke as a non-invasive neuroprotective strategy, which maintained for both short and middle term ischemic reperfusion time. The protective effect is mediated by the modulation of inflammatory response in the ischemic brain.


Introduction
Ischemic stroke is considered to be the third most fatal and disabling disease in the world. At present, the most effective treatment for stroke is intravenous thrombolysis or intravascular interventional treatment within several hours after the onset of stroke. Unfortunately, the proportion of patients who can be treated within a few hours is less than 5%, and even if the infarction is lifted and blood reperfusion is established, the ischemia-reperfusion injury of the brain tissue cannot be ignored, which causes the current unsatisfactory results. Neuroprotective drugs developed over the years have been proven effective in animal models of stroke, but have poor clinical efficacy [1]. Therefore, it is urgent to find auxiliary or alternative treatment to further improve the treatment effect of stroke. In recent years, a variety of remote ischemic preconditioning (RIPC) methods have been tested as feasible treatment strategies for stroke. Our previous studies and other researches have proved that RIPC has a protective effect on stroke in both basic research [2][3][4] and clinical experiments [5][6][7]. RIPC is easy to handle and relatively resistant to reperfusion injury, so it has great clinical advantages. However, for the once delayed non-invasive RIPC method, the duration of protection and mechanisms are still unclear.
Preconditioning is a phenomenon in which the brain protects itself against future injury by adapting to low doses of noxious insults [8]. The concept of cerebral ischemic tolerance was first introduced in the early 1990s. Kitagawa et al. reported the neuroprotective effects against neuronal cell death when adding 2 minutes of transient ischemia 24 hours before global cerebral ischemia in rats [9]. As ischemic conditioning is difficult to realize in the in situ organ, the concept of RIPC is proposed. RIPC is an endogenous protective mechanism through which the short-term sub-lethal ischemia of remote organs can protect the main organs from further severe ischemia. Acute and delayed preconditioning in both heart and brain has different mechanisms. Acute/early preconditioning performed 1 to 3 hours before stroke onset is related to a rapid response such as changes in ion channel permeability and posttranslational modifications of proteins and the protection lasted only several hours.
While delayed preconditioning induced 1 to 7 days before stroke onset induced gene activation and protein synthesis and the protection lasted several days [10][11][12][13]. In most experiments, the protective effects on the brain need hours and sometimes days to fully manifest; thus, delayed preconditioning has been studied as a more effective strategy. To date, studies on the mechanisms of both cardiac and cerebral preconditioning at the molecular, cellular and tissue levels span nearly 30 years [14].
Many studies have shown that the neuroprotective mechanisms of RIPC by a complex cellular regulatory process that involves multiple cellular signaling pathways and leads to enhanced tolerance to ischemia/hypoxia. So far, there are many ways of RIPC, and their effects are not completely consistent. Previous study showed that non-invasive RIPC 5 min ischemia/ 5 min reperfusion for 3 cycles contributed neuroprotection by activating adenosine A1 receptor [15]. Another study showed that 4 cycles of RIPC (5 min/cycle, 40 min total) decreased the expression of neuroinflammation by improving the peripheral immune cell response [2]. Our previous research showed that 3 cycles of RIPC (10 min ischemia/ 10 min reperfusion) significantly reduced infarct size and neuroinflammation by modulating the expression of HIF-1 [4]. Moreover, studies showed that acute (15 min before ischemia), delayed (24 h before ischemia) and chronic (9 d repeated ischemic conditioning) ischemic preconditioning all reduced infarct size in heart following myocardial ischemia [16]. For the 3 cycles of once delayed non-invasive RIPC (10 min ischemia/ 10 min reperfusion), it has been proven effective in our previous study, whereas, the duration of this protection lasting is still unclear.
In this study, we aimed to provide insights into demonstrating how long the protective effect of once delayed non-invasive RIPC can be maintained. First, we tested the infarct size, neurological and behavioral deficiencies at different reperfusion time compared with MCAo and RIPC+MCAo group. Then, we tested the mRNA and protein levels of pro-inflammatory cytokines in the ischemic brain by real-time RT-PCR and ELISA respectively.

Materials and methods
Male Sprague-Dawley (SD) rats were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). The weights were 280-320g. They are placed in a control room at a temperature of 24 ° C and a standard 12-hour light-dark cycle. They can freely obtain food and water and are randomly divided into different groups. The number of animals per group is 12 to 14. All procedures in this study were conducted in accordance with ethical standards, with the Helsinki Declaration,

Middle cerebral artery occlusion (MCAo)
In our experiments, rats were anesthetized with 3-5 % isoflurane in 70 % nitrous oxide and 30 % oxygen, and maintained with 1-3 % isoflurane. For the MCAo model, ischemia and reperfusion was established as previously described [4]. Briefly, the common carotid artery (CCA), right internal carotid artery (ICA), and external carotid artery (ECA) were exposed during the procedure. A silicone-coated nylon suture with a diameter of 0.38 ± 0.02 mm was inserted into the ICA; the silicone-coated nylon suture was within 18-20 mm from the ECA bifurcation to block the MCA, and withdrawed after 90 minutes of occlusion to allow MCA to re-open. Rat rectal temperature was maintained at 37±0.5° C during the entire procedure. The rats in the sham group underwent surgery without MCA occlusion. The cerebral blood flow (CBF) during the surgery before and after occlusion were measured by laser Doppler perfusion monitoring with a laser Doppler probe (PeriFlux System 5000, Perimed AB, Sweden) interfaced to a laptop equipped with the PeriSoft data acquisition software (PeriSoft Systems, Inc., Sweden). The blood gas (PaCO 2 , PaO 2 [mmHg] and pH) and blood sugar (mmol/L) were also examined during the surgery as previously described [17].

Remote ischemic preconditioning (RIPC)
In our experiments, delayed non-invasive RIPC was used, in contrast to the invasive, direct femoral artery occlusion. Briefly, twenty-four hours before MCAo, RIPC was conducted in both hind limbs of rats anesthetized with 1-3 % isoflurane.
Two strip gauze bandages were tied on the two hind limbs simultaneously to occlude blood circulation for 10 minutes, and then released for 10 minutes to allow reperfusion. The occlusion/reperfusion cycle was repeated for 3 times.

Behavioral testing
Behavioral tests were conducted as described previously [4,17]. Behavioral tests were performed to assess rats' neurological function after stroke, including tail hang tests, home cages test and postural reflexes test. All behavioral tests are performed by a person who does not understand the experimental conditions. We trained rats three days before surgery and tested their baseline one day before surgery. All behavioral tests were evaluated before the animals were sacrificed.
For the tail suspension test, hung the tail of the rat about 10 cm from the ground.
Stroke rats will turn to the opposite side (left) of the ischemic hemisphere, and the head will rotate more than 90 °. Each rat was hung for no more than 5 seconds, and each rat was hung a total of 20 times. The percentage of head turns was calculated.
Rats usually used their forelimbs to explore the cage margin. For the home cage limb test, we calculated the number of times when rats' ipsilateral, contralateral, or both forelimbs contacted the cage wall. Instruct the rat to touch the cage wall 20 times. Use the following formula to calculate the percentage of ipsilateral forelimbs used: [ipsilateral + (both /2)] x 100%. In a postural reflex test, rat was placed on a table. We held its tail in one hand, and pushed its shoulder nearly 20 cm for 3 times with the other hand. Non-ischemic rats grasped the table vigorously during the push and scored zero. Rats that had less resistance and became stiff during the referral process received 1 point. If the rat was not resistant, the score was 2.
We also used the Longa scoring system to measure neurological deficits at different times after reperfusion to assess stroke outcomes. The scores were based on the following criteria: 0 = no defect, 1 = inability to stretch the left front foot, 2 = circle left 3 = Fall to the left, 4 = Unable to walk away and lose consciousness, 5 = Death.

Infarct size measurement-TTC staining
For 6 and 72 hours reperfusion animals, the infarct area was measured using 2, 3, 5-triphenyl-2H-tetrazolium chloride (TTC) staining. Measure non-ischemic hemispheres and non-ischemic region and calculate infarct area according to the following formula: [(area of the non-ischemic hemisphere -area of the non-ischemic region in the ischemic hemisphere)/area of the non-ischemic hemisphere] × 100%. Detailed protocols have been described previously.

Infarct size measurement-Cresyl violet staining
For the long-term reperfusion induced by stroke, the infarct volume was measured by Cresyl violet (CV) staining as the TTC staining method does not reflect the infarct size clearly. Animals were anesthetized and transcardially perfused with The slides were washed twice with ddH 2 O and immersed in 95% ethanol for 2 minutes. Then, they were cleared twice for 5 min with xylene, sealed with neutral gum, and finally observed under microscope.

Quantitative RT-PCR analysis
To measure TNF, IL-1 and IL-6 mRNA expression, total RNA was isolated from the ischemic brain, which was collected on ice and stored at −80℃ immediately after the animals were euthanized. RNA was extracted with TRIzol reagent ( for 5 min, 45 cycles at 95℃ for 5 s, 60℃ for 5 s, and 72℃ for 10 s, followed by 72℃ for 1 min.

ELISA for quantifying pro-inflammatory cytokines
We measured 3 pro-inflammatory cytokines: TNF, IL-1 and IL-6 using ELISA The number of rats in each group was 12-14.

RIPC reduced infarct size at short reperfusion time following stroke
To test whether once non-invasive delayed RIPC was neuroprotective in different reperfusion time including 6 h, 72 h and 7 d. We measured the weight and infarct size by TTC and CV staining. Firstly, we detected the CBF levels, blood sugar and blood gas between groups which were the requirement of stroke model. CBF levels were monitored during MCAo surgery, and there was no difference between groups. It was reduced nearly 78% of baseline during ischemia and reestablished to 80% of baseline following reperfusion (Fig. 1B). At the same time, there were no differences of blood sugar and blood gas between groups, either ( Fig. 1C and D). Results showed that RIPC significantly attenuated the weight loss at 72 h post-stroke (Fig. 2D), while RIPC had no effect of the weight loss at other reperfusion time ( Fig. 2A and G). TTC staining results showed that RIPC significantly reduced infarct size from 39.26 ± 1.51 to 31.31 ± 1.68 after reperfusion 6 h ( Fig. 2B and C). Similarly, RIPC significantly reduced infarct size from 47.11 ± 1.14 to 36.44 ± 1.82 at 72 h post-stroke ( Fig. 2E and F). Whereas, CV staining results showed that there was no significant difference of the infarct size receiving RIPC compared with MCAo group at 7 d post-stroke ( Fig.   2H and I).

RIPC improved neurological and behavioral function at early stroke
After validating that once non-invasive delayed RIPC reduced infarct size at acute and middle term ischemic reperfusion time following stroke, we further examined the neurological score and behavioral performance receiving RIPC. The results showed that RIPC significantly attenuated neurological dysfunction at 6 and 72 h post-stroke, while there's no significant change at reperfusion 7 d after MCAo (Fig. 3A). Such protection of RIPC in these reperfusion time-points were also observed in the behavioral performance test especially in the tail hang test and home cage test, in which RIPC significantly improved the behavioral performance at 6 and 72 h post-stroke ( Fig. 3B and C). There was no significant difference in the postural reflex test at any of the reperfusion time-point, but we also observed a decrease trend in RIPC group (Fig. 3D).

RIPC down-regulated pro-inflammatory factors in the ischemic brain at short-term ischemic reperfusion time following stroke
To test how the inflammatory status was regulated by RIPC at different ). Whereas, no any significant changes of IL-1 expression were observed at 7 d following MCAo (Fig. 4B and 5B). Finally, it was showed that RIPC down-regulated the expression of IL-6 on the middle-term of reperfusion time (72 h post-stroke) ( Fig.   4C and 5C).

Discussion
In the present study, we investigated the neuroprotection of once delayed non- in myocardial ischemia [12]. They indicated that ischemic preconditioning was a short period of ischemia, which did not affect the ischemic tissue, but had a protective effect on subsequent, prolonged ischemia. While Kitagawa et al. first introduced cerebral ischemic tolerance in the early 1990s. They discovered 2 min of transient ischemia 24 h before global cerebral ischemia had a neuroprotective effect against neuronal cell death [9,18]. Researchers regarded ischemic preconditioning as a powerful tool in understanding the endogenous mechanisms by which the ischemic organs are protected [19]. In terms of clinical applicability for myocardial infarction and stroke treatment, RIPC had advantages over conventional ischemic preconditioning by reducing the higher risk directly to the ischemic organ [20,21].
RIPC refers to a repeated transient ischemia/ reperfusion in a remote organ to prevent prolonged ischemia of other vital organ, which now was widely used in the protection of heart and brain ischemia. In contract to invasive RIPC, we mainly focus on noninvasive method which is often established by tourniquet or strip gauze bandages. The important findings of our study were that once delayed non-invasive RIPC (3 cycles of 10 min ischemia/ 10 min reperfusion) reduced infarct size, attenuated the loss of neurological function and behavioral performance only at acute (6 h) and middle-term (72 h) reperfusion time, but not long-term (7 d) (Figs 2 and 3). The results were consistent with our previous study, in which we have demonstrated that RIPC reduced ischemic/ reperfusion injury at 48 h post-stroke [4]. 3 but not 7 d of reperfusion following global ischemia [22]. Although different ischemic preconditioning positions were used, these results supported that transient ischemic preconditioning treatment could not maintain long-term protection.
However, other studies showed that once rapid non-invasive RIPC (3 cycles of 15 min ischemia/ 15 min reperfusion) had chronic protective effect against distal MCAo even after 60 d [3]. And these contradictory conclusions can be explained by the different model chosen for the two experiments. Moreover, these results supported that RIPC was more effective to improve the infarct in the cortex rather than basal ganglia injury Inflammatory response plays an important role in the pathogenesis of ischemic stroke. A large number of studies have shown that neuroinflammatory response is involved in the prognosis of cerebral ischemia-reperfusion injury [23,24]. Therefore, in theory, inhibiting the inflammatory response after stroke can reduce stroke injury and improve the clinical prognosis. Previous studies have shown that RIPC reduced systemic neuroinflammatory response [2][3][4]15]. To examine whether reducing infarction was influenced by the elimination of inflammation in the ischemic brain, we then measured the levels of neuroinflammation in the ischemic brain. We found that RIPC reduced the mRNA and protein levels of pro-inflammatory cytokines, including TNF, IL-1 and IL-6 at acute and middle terms of reperfusion in the ischemic brain, but had no effect on long term reperfusion (Figs 4 and 5). The decrease of cytokines release is consistent with the reduction of the infarct size, suggesting that once delayed non-invasive RIPC improved the regional ischemia by reducing the expression of neuroinflammatory response in the ischemic brain after In fact, RIPC has been moved into clinical trials for several years and it has been proven to be effective in the prevention or treatment of cerebrovascular disease [5][6][7]20]. The importance of our study was to confirm the limited therapeutic time window of RIPC we used. For different ischemic preconditioning methods and stroke models, RIPC may have different protective effect. Only known that how long it works, can we explore the mechanism and complete further clinical transformation.
There are also several limitations in the present study. First of all, inhibitors of inflammatory cytokines need to be used to demonstrate the interaction between brain injury and inflammatory response in the future study. Secondly, the mechanism of RIPC should be further explored, such as whether the hypothalamus-pituitary-adrenal axis is involved in the protective effect of RIPC. Last but not the least, RIPC was used only once in our experiment, and the parameters of RIPC worth studying to obtain a chronic protective effects for further clinical application.

Conclusion
In this study, we provided strong evidence that once delayed non-invasive RIPC protects against stroke as a non-invasive neuroprotective strategy which just at short and middle term ischemic reperfusion time. The protective effect was mediated by the modulation of inflammatory response in the ischemic brain.

Acknowledgments:
We thank Xuan Liu and Haiteng Ji for their long-term feeding of experimental