|Year : 2015 | Volume
| Issue : 2 | Page : 159-166
Neural transmission pathways are involved in the neuroprotection induced by post- but not perischemic limb remote conditioning
Changhong Ren1, Kaiyin Liu2, Ning Li1, Xiaowen Cui3, Jinhuan Gao1, Xunming Ji1, Yuchuan Ding2
1 Institute of Hypoxia Medicine, Xuanwu Hospital, Capital Medical University; Center of Stroke, Beijing Institute for Brain Disorder; Beijing Key Laboratory of Hypoxia Translational Medicine, Beijing, China
2 Department of Neurosurgery, Wayne State University School of Medicine, Detroit, Michigan, USA
3 Institute of Hypoxia Medicine, Xuanwu Hospital, Capital Medical University, Beijing, China
|Date of Submission||08-Oct-2015|
|Date of Acceptance||26-Nov-2015|
|Date of Web Publication||31-Dec-2015|
Institute of Hypoxia Medicine, Capital Medical University, Xuanwu Hospital, Chang Chun Road 45, Beijing - 100 053
Source of Support: None, Conflict of Interest: None
Background: Remote ischemic preconditioning (PreC) and postconditioning (PostC) have all been shown to be neuroprotective against ischemia/reperfusion (I/R) injury. However, the underlying mechanisms of ischemic perconditioning (PerC) remain largely unknown. This study aimed to investigate the potential role of neural transmission pathways in the transference of protective signals evoked by PerC.
Materials and Methods: Male Sprague-Dawley rats were randomly allocated into 12 groups [sham, middle cerebral artery occlusion (MCAO), MCAO+PerC, MCAO+PerC+vehicle, MCAO+PerC+Capsaicin, MCAO+PerC+sham, MCAO+PerC+denervation, MCAO+PostC, MCAO+PostC+vehicle, MCAO+PostC+sham, MCAO+PostC+Capsaicin, MCAO+PerC+denervation]. The I/R model was established by 90-min occlusion of the right middle cerebral artery and subsequent 24 h reperfusion. Remote conditioning was induced with three cycles of 10 min ischemia/10 min reperfusion of the femoral arteries bilaterally. Nerve block was conducted by local capsaicin treatment of exposed nerves or femoral and sciatic nerve transection. Cerebral infarct volumes were quantified by 2, 3, 4-triphenytetrazolium-chloride stain assay. The phosphorylation of Akt was detected by Western blot.
Results: Remote ischemic PerC and PostC therapies reduced the infarct size and attenuated neurological deficits. Blocking the neural transmission pathways abolished the protective effect of PostC but had no effect on PerC. Further, blocking the neural transmission pathways reduced periinfarct Akt activation of PostC but had no effect on PerC.
Conclusion: Unlike PostC, neural transmission pathways may not play a significant role in the transference of PerC-induced neuroprotection after I/R injury.
Keywords: Ischemia, neural pathway, remote ischemic per conditioning, remote ischemic postconditioning, reperfusion injury
|How to cite this article:|
Ren C, Liu K, Li N, Cui X, Gao J, Ji X, Ding Y. Neural transmission pathways are involved in the neuroprotection induced by post- but not perischemic limb remote conditioning. Brain Circ 2015;1:159-66
|How to cite this URL:|
Ren C, Liu K, Li N, Cui X, Gao J, Ji X, Ding Y. Neural transmission pathways are involved in the neuroprotection induced by post- but not perischemic limb remote conditioning. Brain Circ [serial online] 2015 [cited 2020 Oct 23];1:159-66. Available from: http://www.braincirculation.org/text.asp?2015/1/2/159/172897
Changhong Ren∗, Kaiyin Liu∗
| Introduction|| |
Ischemic stroke is caused by an interruption of cerebral blood flow to areas of the brain, which affects neuronal function. Stroke represents the fourth most common cause of death and one of the leading causes of disability in the US.  Thus, there is a strong impetus to come up with novel therapeutic options for the treatment of ischemic stroke to reduce mortality and morbidity.
Ischemic conditioning refers to the application of short, nonlethal episodes of ischemia to a target organ or tissue to confer powerful protection against a sustained, lethal episode of ischemia. This broad term of ischemic conditioning is further divided by when the ischemic stimulus is applied into ischemic preconditioning (PreC) (ischemic stimulus applied before vessel occlusion), ischemic perconditioning (PerC) (ischemic stimulus applied during vessel occlusion) and postconditioning (PostC) (ischemic stimulus applied during vessel reperfusion). Remote ischemic conditioning is the stimulation of a distant, nonvital organ to elicit protection to a vital organ such as the heart or brain.
Of the previously described ischemic conditioning methods, PreC is one of the most well-studied. , Remote ischemic PreC has been studied clinically where it has shown efficacy in providing neuroprotection. , In contrast to ischemic PreC, ischemic PostC is a relatively new concept.  Both PerC and PostC are potentially more useful for clinical translation. Several experimental and clinical studies have shown that strong neuroprotection can be elicited using limb PerC and PostC. ,,,,, Despite the wealth of molecular biology data detailing the protective mechanisms of remote ischemic conditioning, how the protection can be transferred from the limb to the target organ remains unknown.  Some proposed mechanisms include: Humoral factors carried through the systemic circulation, peripheral and autonomic nervous transmission, and leukocyte-mediated transmission.  Wei et al. have shown that the neuroprotective mechanism of limb remote PreC using hind limbs was transferred through sensory afferent nerves from the preconditioned limb to the brain.  It was shown in their study that the neuroprotective effects of limb PreC was abolished when sensory nerve inhibitor, capsaicin, and ganglion blocker hexamethonium were applied to the animals.  Further, Xiao et al. have shown that the electrical stimulation of peripheral nerve in PostC resulted in neuroprotection in a rat distal middle cerebral artery occlusion (MCAO) model.  Ren et al. also reported that capsaicin abolished the neuroprotection of PostC in a rat ischemic stroke model.  Taken together, peripheral nerve transmission may play an important role in the transference of protection. In the present study, we wanted to investigate and compare the possible mechanism of neuroprotection transference from the limb to the brain in remote PerC and PostC. To answer these questions, we used a rat model of remote conditioning and Western immunoblotting to narrow down the mechanism of limb remote conditioning.
| Materials and Methods|| |
All animal experiments were approved by the Animal Care and Use Committee of Xuanwu Hospital, Capital Medical University, China, and conducted according to the National Institutes of Health guidelines. A total of 90 adult male Sprague-Dawley rats (280-320 g) were used in this study (Vital River Laboratories, Beijing, China). The animals were maintained on a 12-h light/dark cycle with unlimited access to food and water.
Male Sprague-Dawley rats were randomly allocated into 12 groups [Figure 1]: Sham, MCAO, MCAO+PerC, MCAO+PerC+vehicle, MCAO+PerC+Capsaicin, MCAO+PerC+sham, MCAO+PerC+denervation, MCAO+PostC, MCAO+PostC+vehicle, MCAO+PostC+sham, MCAO+PostC+Capsaicin, and MCAO+PerC+denervation.
|Figure 1: The representative sketches of experiment groups. Cap = Capsaicin, NR = Nerve resection|
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Focal cerebral ischemia
1.5-3.5% enflurane in 70% nitrous oxide and 30% oxygen (Bickford veterinary anesthesia equipment model no. 61010; AM Bickford Inc., Wales Center, NY, USA) was used for anesthesia induction and maintenance. Focal cerebral ischemia was generated using the intraluminal MCAO model as described previously. , Briefly, the right common carotid artery and the right external carotid artery (ECA) were exposed. The ECA was then dissected distally, ligated, and coagulated. The MCA was occluded using a heparinized intraluminal ﬁlament (diameter 0.28 mm) (Shadong Inc, Beijing, China). After 90 min, the suture was withdrawn. During the operation, rectal temperature was monitored and maintained at 37 ± 0.5 ° C with a thermostat-controlled heating blanket (Biotech-China Inc, Shanghai, China). Sham-operated rats underwent an identical surgery except that the MCA was not occluded.
Remote ischemic per- and postconditioning
Under enflurane anesthesia, bilateral femoral arteries were exposed before middle cerebral artery (MCA) occlusion. At 30 min after ischemia onset, remote PerC was conducted in the bilateral lower limbs by occluding and releasing the femoral artery for three cycles; each occlusion or release lasted for 10 min [Figure 1]a. The bilateral femoral arteries in the control ischemic group were separated and exposed as was done in the remote PerC group but without occlusion and release. Remote PostC was initiated at 30 min after reperfusion. The protocol for PostC was the same as the PerC treatment described above. A second person who did not perform surgery randomly assigned the rats into different groups. Since enflurane might be neuroprotective,  the same period of enflurane administration was applied to the rats who were controls.
Infarct size measurement
Infarct size was measured according to previous methods.  Twenty-four hours after surgery, the rats were anesthetized with 1% chloral hydrate, and then the brains were removed and sectioned coronally at the level of optic chiasm at 2-mm intervals, generating a total of six sections, which were stained with 2% solution of 2, 3, 4-triphenytetrazolium-chloride (TTC). Using a computerized image analysis system (Image-Pro Plus, version 5.1, Media Lybernetics, Silver Spring, MD, USA), the area of infarction was defined at the sides of the inner section. Infarct size of the ischemic region was normalized to the nonischemic region and expressed as a percentage, and an average value from the six slices was presented.
Neurological deficit was determined through the neurobehavioral scoring system developed by Belayev et al.  with some modifications. The scoring system was graded on a scale of 0 to 12 (minimal score: 0; maximal score: 12). The tests included: (1) postural reflex test to examine upper body posture, and (2) the forelimb placing test to examine sensorimotor integration. Thirteen rats were used for each group.
Drug injection and surgery for denervation
To test whether the neural pathway is involved in transferring protective signaling from the limb to the brain during remote conditioning, the afferent nerve blocker capsaicin, was used to study the potential protective mechanisms of remote PerC and PostC. The treatment method was performed as described previously.  Briefly, capsaicin was dissolved in 10% ethanol, 10% Tween-80 (Beijing Chemical Works, Beijing, China) and 80% saline to a final concentration of 1%. This solution was then applied to the bilateral femoral nerves of the rats. In brief, the rats were anesthetized with enflurane and the left and right femoral nerves were exposed. Approximately 1-cm long segments of the nerves were isolated with Parafilm (Beijing Chemical Works, Beijing, China) and small pieces of capsaicin solution-moistened gelfoam were wrapped around the nerve for 30 min. The capsaicin-treated areas were then flushed with saline and the wound was closed. In the control animals, the bilateral femoral nerves were similarly treated with saline. PerC, PostC, and focal ischemia were performed 4 days later. The operator performing the surgery was blind to the rats' conditions. To further examine whether the nerve pathway was involved in the neuroprotection of remote conditioning, sciatic and femoral nerve denervation were performed as reported previously.  Briefly, under enflurane anesthesia, bilateral sciatic nerve (SN) and femoral nerve (FN) were exposed. Approximately 0.5-cm segments of the FN and SN were resected followed by a recovery period of 20 min. Focal ischemia was performed 2 h after the nerve transection. In the control animals, only the nerves were exposed. The operator performing the surgery was blind to the rats' conditions.
Western blot analysis was used to assess phosphorylated Akt (p-Akt) expression. Protein was isolated from the rat periinfarct region at 1 day, 7 days, and 14 days after reperfusion. Protein (40 μg) was electrophoresed on 12.5% SDS polyacrylamide gels, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA). The membrane was probed with rabbit polyclonal anti-phospho-Akt (Ser473) antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA). The membrane was then washed three times and reincubated with a secondary antibody. An enhanced chemiluminescence (ECL) system was used to detect immunoreactive bands by chemiluminescence (GE Healthcare, Buckinghamshire, UK). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to verify equal loading. Quantification of protein amounts were performed using Image-Pro Plus software 5.0 (Rockville, MD, USA).
Statistical analyses were made with Statistical Package for the Social Sciences (SPSS) version 17.0 software (IBM Corporation, Armonk, NY, USA). One-way analysis of variances with Bonferroni corrected post hoc correction was used to assess differences between groups. Values were expressed as mean standard deviation. P < 0.05 was considered to be statistically significant.
| Results|| |
Remote ischemic perconditioning and postconditioning conditioning therapies reduced the infarct size and attenuated neurological deficiency
Brain infarct volume was detected 24 h after reperfusion. The volume of infarcted tissue found in the six brain slices was calculated as a percentage of the contralateral hemisphere in rats. Those rats that were part of the MCAO+PerC (n = 8) group showed 16.6 ± 2.9% infarct size compared to a 34.6 ± 3.8% infarct size in the MCAO group (n = 6) [Figure 2]a. The reduction of 52% proved to be significant (P < 0.001). The rats in MCAO+PostC group (n = 7) showed 18.4 ± 2.4% infarct size [Figure 2]a. Similarly, this reduction by 47% proved to be significant (P < 0.001). There were no significant differences between MCAO+PerC group and MCAO+PostC group (P > 0.05). After the occlusion and release were conducted, cyanosis and other potential complications in the lower limbs were not observed.
|Figure 2: Remote ischemic conditioning therapy reduced ischemic injury. (a) Remote limb ischemic conditioning reduced infarct size measured at 24 h after reperfusion. Bar graphs show the average infarct size of the six brain slices (b) Remote limb ischemic conditioning attenuated neurological deficiency. Neurological deficits were determined using neurobehavioral scoring system (higher scores correspond with more severe deficits) *P < 0.05, versus MCAO group, n = 13 per group ***P < 0.001, versus MCAO group|
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Next, we analyzed the effect of PerC or PostC treatment on the neurological functional outcome. As shown in [Figure 2]b neurological deficits including body posture and sensorimotor integration were significantly improved at 24 h after reperfusion in the MCAO+PerC group (n = 13 (5.3 ± 1.4) compared with the MCAO group (n = 13) (6.61 ± 0.9) (P < 0.05) [Figure 2]b. The rats in MCAO+PostC treatment (n = 13) (5.07 ± 1.4) also attenuated the neurological deficiency as compared with the MCAO group (P < 0.05) [Figure 2]b. There were no significant differences between the MCAO+PerC group and MCAO+PostC group (P > 0.05).
Blocking the nerve pathways inhibited the protective effect of postconditioning but had no effect on perconditioning
We next examined whether the afferent nerve pathways transfer the protective signaling from the ischemic conditioned limb to the brain. First, local application of capsaicin to the FN increased the infarct size in rats receiving MCAO+PostC treatment (19.4 ± 3.8% versus 32.6 ± 4.4%; P < 0.01) [Figure 3]a and b but did not increase the infarct size in rats receiving MCAO+PerC (17.4 ± 4.3% versus 18.2 ± 5.9%; P > 0.05) [Figure 3]a and b. To further block the nerve pathway, we performed the denervation surgery; bilateral SN and FN transection increased the infarct size in rats receiving MCAO+PostC treatment (15.8 ± 2.3% versus 29.3 ± 5.5%, P < 0.01) [Figure 3]c and d; however, the denervation still had no effect on the infarct size of the rats receiving MCAO+PerC (17.3 ± 4.5% versus 16.6 ± 5.3%, P > 0.05) [Figure 3]c and d. These results suggest that neural activity contributes to the protective effects of PostC but had no effect on PerC.
|Figure 3: Blocking the nerve pathways abolished neuroprotection of PostC but had no effect on PerC. (a) Representative infarcts stained by TTC from each group (b) Bar graphs showed the effect of capsaicin on infarct size in each group (c) Representative infarcts stained by TTC from each group (d) Bar graphs showed the effect of bilateral sciatic and femoral nerve transection on infarct size in each group (e) The effects of capsaicin on neurological function. n = 12 per group (f) The effect of bilateral sciatic and femoral nerve transection on neurological function. n = 12 per group. Cap, capsaicin *P < 0.05, **P < 0.01|
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Next, we analyzed the effects of capsaicin and nerve transection in remote ischemic conditioning on the neurobehavioural score. First, local application of capsaicin to the FN deteriorated the neurological function in rats receiving MCAO+PostC treatment (7.1 ± 1.3 versus 5.1 ± 1.5 P < 0.05). [Figure 3]e but did not deteriorate the neurological function in rats receiving MCAO+PerC (5.8 ± 2.1 versus 5.2 ± 1.9; P > 0.05) [Figure 3]e. Bilateral SN and FN transections deteriorated the neurological function in rats receiving MCAO+PostC treatment (7.1 ± 1.2 versus 5.9 ± 1.1, P < 0.05) [Figure 3]f; however, the denervation still had no effect on the infarct size of the rats receiving MCAO+PerC (5.9 ± 1.3 versus 5.6 ± 1.4, P > 0.05) [Figure 3]f.
Blocking the nerve pathways reduced periinfarct phosphorylated Akt expression in MCAO+PostC but had no effect on MCAO+PerC
Next, we further analyzed the expression of p-Akt which is a prosurvival signal after remote ischemic conditioning treatment. All the MCAO groups had significantly lower levels of p-Akt when compared with sham at 24 h after reperfusion (P < 0.01) [Figure 4]. The expression of P-Akt was upregulated by both the PerC and PostC (P < 0.05) [Figure 4]. Local application of capsaicin to the FN blocked upregulation of p-Akt expression induced by PostC (P < 0.05) but had no effect on PerC [Figure 4]a. Similarly, bilateral SN and FN transection blocked upregulation of p-Akt expression induced by PostC (P < 0.05) but had no effect on PerC (P > 0.05) [Figure 4]b.
|Figure 4: p-Akt (Ser473) level, as detected by immunoblot analysis, is increased after nerve severance after PostC but had no effect on PerC. (a) Local application of capsaicin to the femoral nerve reduced the Akt activation after PostC but had no effect on PerC. Upper panel shows the representative immunoblot image. Bottom panel shows the densitometric analysis (b) Bilateral sciatic and femoral nerve transections reduced the Akt activation of PostC but had no effect on PerC. Bottom panel shows the densitometric analysis. Data are presented as mean ± SD (N = 6), *P < 0.05, **P < 0.01|
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| Discussion|| |
Using evidence from a rat cerebral ischemic/reperfusion (I/R) model, this study indicated that remote conditioning after transient I/R in bilateral hind limbs (PerC and PostC) can induce definitive neuroprotection characterized by reduced infarct size and attenuated neurological deficits. The potential of neuroprotection was almost equivalent between the PerC and PostC groups. It is plausible that remote PerC transferred protection to the ischemic brain through a mechanism independent of afferent nerve activity since inhibition of nerve activity through direct block and transection did not abolish its protection. In contrast, inhibition of nerve activity abolished PostC neuroprotection, which was consistent with the data reported previously by Ren et al. 
Ischemic conditioning targets ischemia/reperfusion injury
Depending on the temporal relation between sublethal ischemia (conditioning) and lethal ischemia (vessel occlusion), the ischemic stimulus can be applied before ischemia (PreC), during ischemia, (PerC) and after reperfusion (PostC).  However, the onset of acute ischemic stroke is often unpredictable, thus limiting the clinical applicability of PerC . In order to be more clinically relevant, less invasive methods for applying remote ischemic PerC and PostC are required.  We reported previously that the application of PerC immediately at the beginning of MCAO had a neuroprotective role against I/R injury.  In this current study, we purposefully delivered the PerC therapy at the end of the index ischemia period to better mimic the clinical scenario. We found that this PerC therapy still had significant neuroprotection as indicated by decreased infarct volume and improved neurological score. Using this rat I/R model, application of PostC at 30 min after reperfusion similarly reduced I/R injury. In contrast to our result, Qi et al. reported that the application of PostC at 30 min after reperfusion did not reduce the infarct size. Since the neuroprotection is dependent on the time of application,  the contrasting results are possibly due to differences in which the remote conditioning was applied. In fact, using another I/R rat model where three cycles of 15 min ischemia/15 min reperfusion was performed, Ren et al. reported that PostC could reduce infarct size even at 6 h after reperfusion.  Nevertheless, it remains unclear whether more cycles of either PerC or PostC remote conditioning or a combination both methods would lead to greater protection. A recent report from our group suggested that repetitive PostC offered an alternative to increase the protective effects after PerC, which laid the foundation for further clinical translation of PerC and PostC combination treatment. 
Remote limb perconditioning neuroprotection may be transferred independent of neural pathways
Our knowledge on the complex mechanisms contributing to remote PerC treatment in neuroprotection is still full of obscure details. Recently, Szijΰrto et al.  and Hess et al.  reviewed the underlying mechanisms of PerC and PostC. According to their theory, three main mechanisms, namely, humoral, neural, and systemic hypothetically interact with each other to provide the target organ protection from a remote site. However, this categorization cannot be applied directly to PerC owing to the lack of sufficient experimental data. To the best of our knowledge, no data have been reported so far, which investigated the role of neural elements behind the neuroprotective effects of PerC.  Czigΰny et al.  published that the hepatoprotective effects evoked by hind limb remote ischemic PerC were completely abolished after transection of the ipsilateral FN and SN. Wei et al. reported that local application of capsaicin to the FN abolished the neuroprotection offered by limb remote ischemic PreC.  Based on these data, we aimed to investigate whether neural elements were involved in the transference of neuroprotection of PerC against cerebral I/R injury. In this study, we first blocked the afferent nerves with local capsaicin treatment. Our result showed that capsaicin did not abolish the protective effect of remote PerC against I/R injury but did abolish PostC neuroprotection as corroborated by Ren et al.  as a control. We speculated that the capsaicin treatment on the FN could not fully block the activity of afferent neurons. Next, we performed transection of the bilateral FN and SN. The denervation still did not abolish the neuroprotection of remote PerC. However, as expected, the denervation treatment reversed neuroprotection offered by remote PostC. Our results suggested that the afferent nerve pathways may serve as a connection between the remote organ or limb and the ischemic brain in PostC but not in PerC. In the light of Czigàny et al., this observation also underscores the fact that remote PerC protection may be transferred through different mechanisms to different target organs.  To further determine whether the other mechanisms (i.e., the humoral and systemic pathway) are implicated in the neuroprotection offered by remote ischemic PerC, further experiments should be performed.
Akt pathway may be an important downstream mediator of neuroprotection in remote conditioning
During reperfusion, the downstream effects of multiple mechanisms appear to converge on the upregulation of reperfusion injury salvage kinase pathway.  PI3K/Akt is a key prosurvival signal after experimental stroke and ischemic conditioning treatment.  Hoda et al.  reported that limb remote ischemic PerC upregulated p-Akt compared with the embolic MCAO rat. Considering the determinative role of this pathophysiological molecular pathway, we attempted to quantify changes in p-Akt expression in our I/R model. In this study, we found that both remote PerC and PostC upregulated p-Akt compared with MCAO control rats, which are in accordance with the findings from Hoda et al. Further studies showed that blocking the nerve pathways reduced the p-Akt activation of MCAO+PostC in the periinfarction region but had no effect on PerC. This result suggested that the triggers of Akt phosphorylation transfer from the periphery to the central nervous system through neural pathways for PostC but not PerC. Further experiments may be conducted with specific Akt kinase inhibitors to solidify this link as well as search for additional neuroprotective pathways activated by remote limb conditioning.
| Conclusion|| |
In conclusion, our study has shown that the neuroprotective effect of PostC is indeed transferred through neural pathways from the periphery to the brain, as shown by many previously. On the other hand, we have shown for the first time that the neuroprotective signals evoked by PerC are transferred to the brain independent of the neural pathways. Further experiments are underway to determine alternate mechanisms through which PerC neuroprotection may be transferred as well as the molecular details of this protection. The nonduplicity of neuroprotective mechanisms of PerC and PostC may open the door to novel additive or synergistic combination approaches in the treatment of ischemic stroke.
This study was supported in part by the National Natural Science Foundation of China (no. 81573867) and Projects in the National Science and Technology Pillar Program during the 12th 5-year plan period (No, 2013BAI07B00).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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