| BackgroundIschemic stroke means the blood supply disorder of local cerebral tissue caused by various reasons. It leads to ischemia and hypoxia necrosis, and then produce the corresponding clinical neurological deficits. The disease brings a heavy burden to the society and family because of its high incidence, high mortality, high morbidity, high recurrence rate and complications. In recent years, Ischemic stroke is becoming a major disease which threats to human life. Thus, to find effective treatments and explain its mechanism has been the hot topics in the study of the medical profession.As known more than 50% vessels can spontaneously recanalize after cerebral infarction. A series of complex pathophysiological changes happened when blood recanalized. This eventually lead to neuronal cell death and late-onset neural cell death, also improve the neurological function and the formation of cerebral infarction after cerebral ischemia reperfusion injury. Medical calls it Cerebral Ischemia Reperfusion (CIR) damage. Its mechanism is relatively complex, which is related to free radical formation, toxicity of excitatory amino acids intracellular calcium overload, inflammatory reaction and abnormal gene expression and so on [1]. A large number of literature reports mitogen-activated protein kinase (MAPK) cascade pathway participates in the damage or repair of nerve cells after CIR [2-5]. It plays a key role in the signal transduction of apoptosis. Now, at least six members of the MAPK have been confirmed. Among them, the followings are studied more.1) extracellular signal regulated kinase(ERK) pathways:responses to tyrosine kinase receptor, G protein coupled receptors, growth factor receptor; 2) stress-activated protein kinase(SAPK/JNK) pathways:responses to the TNFa, IL1 and thermal reaction; 3) p38 mitogen-activated protein kinase(p38MAPK) pathway:responses to the heat shock reaction, TNFα、IL-1. These three major signaling pathways are all activated in neurons and astrocytes when cerebral ischemia reperfusion happens [6].Clinical reports and experimental studies have confirmed the effect of acupuncture on Ischemic stroke. ERK signal pathway, JNK signal pathway and p38MAPK signal pathway were studied in the early stage by our research group and some achievements were obtained. Through testing the expression of p-ERK、p-JNK、 p-p38MAPK, we found that electro acupuncture can increase the expression level of p-ERK, can also downregulate the expression level of p-JNK, p-38MAPK. It is speculated that electro acupuncture treatment on cerebral ischemia reperfusion injury may be related to the activation of ERK signaling pathway, and may also be related to the inhibition of JNK signaling pathway and p38MAPK signaling pathway. And to a certain extent, it provides a basis for the hypothesis that "the stimulation of acupuncture-complex signal control-Benign repair mechanism to start is a key mechanism for the treatment of cerebral ischemia reperfusion injury". However its mechanism is not yet clear. And according to the current review of the literature, immunohistochemistry, western blot, RT-PCR were widely used for the detection of related factors of the family signaling pathways. These methods are relatively mature. However, there are some limitations. Double immunofluorescence method can mark two kinds of proteins in the same cell or tissue. In addition, confocal laser scanning microscopy (CLSM) as one of the Nova biomedical laboratory research instrument is being applied in medical research because of its higher image sharpness, stronger index observation sensitivity, and more objective, accurate data acquisition technology. As a result, this topic is proposed to explore possible mechanisms of electric acupuncture treatment on cerebral ischemia reperfusion injury through observing the expression of MAPK signal pathway in the brain by using CLSM and double immunofluorescence method.ObjectiveThrough observing the effect of electro acupuncture on neurologic deficits, focal cerebral infarction volume and expression of p-ERK/p-JNK/p-p38MAPK in rats after ischemia reperfusion, exploring possible mechanisms of electric acupuncture treatment on cerebral ischemia reperfusion injury. Hope to provide scientific and theoretical basis for clinical treatment.MethodsAnimal grouping:150 rats are randomly divided into three groups which are sham group, model (IR) group and electric acupuncture (EA) group. Each group is divided into five subgroups, namely 2 h,6 h,1 d,3 d,7 d, a total of 15 groups, each subgroup 10 rats.Model preparation:Middle cerebral artery occlusion (MCAO) was used for establishing IR model using Longa’s modified method. The rats were fasted for 12h prior to surgery but were given free access to water. The detailed operation was made as follows:rats were anesthetized with intraperitoneal injection of 10% chloral hydrate (0.35mL/100g); 2cm longitudinal cut was made on the skin at 0.3cm of the left cervical anterior midline. The left common carotid artery (CCA), external carotid artery(ECA), internal carotid artery (ICA), and vagus nerve were exposed. Thread was inserted through CCA, ICA, and ECA, but without ligation; ligation of the CCA and ECA was made proximal to the heart; ligation of ICA was temporarily closed by a microvascular clamp; a small cut at 3mm at the branch of ICA and ECA was made. A monofilament nylon suture wasinserted (1.8±0.5cm)from the CCA into the ICA until it was blocked by the clamp; the clamp was loosened and the suture was rapidly inserted further into the ICA. The angle of insertion of the suture was adjusted and the CCA was gently pulled to make the suture enter the brain. When the depth of the suture was approximately 1.8 ± 0.5cm (calculated from the branch of the blood vessel) and slight resistance is observed during insertion, slight resistance ensures that the suture, inserted in the MCA and MCAO, is successfully established. The ICA was tightly ligated and the wound was sutured. Reperfusion was made 30min after MCAO: the suture was gently pulled back to the CCA, and the rest of the suture was cut off. Animals in the sham group were subjected to the same surgical procedures, except that they were exposed only to MCA, but without occlusion with insertion of nylon suture into the MCA. Rats were kept in separate cages for observation after the wound was sutured and were sterilized with povidone-iodine tampons.Electric acupuncture treatment method:After model preparation, different treatments were done to different groups. EA group:Selecting "Chize(LU 5)& Hegu"(LI 4) and "Zusanli(ST 36)&Sanyinjiao"(SP 6). Keeping 20 min each time with dilatational wave,5-10 Hz frequency, voltage 3-5V. Rats in 2h,6h, Id subgroups were treated only once, while 3d and 7d subgroups were treated each day. Rats in model group and inhibitors group were grabbed to fixed only, without electric acupuncture treatment at the same time each day.Observation index:Neurological deficits, cerebral infarction volume, the expression level of p-ERK, p-JNK、p-p38MAPK.Data processing:All the data statistics are conducted on SPSS20.0. Data were expressed as mean ± standard deviation (x±s). One-way ANOVA and LSD were used to analyze all experimental data. P< 0.05 means difference was statistically significant.ResultsNeurological deficit scores:The neurobehavioral performance of rats in Sham group were not seen unnormal. And the Neurological deficit scores were zero. While the neurobehavioral performance of rats in IR group and EA group appeared different degree of abnormal behavior with 2-3 Neurological deficit scores. Compared with sham group, the differences of IR group and EA group show statistical significance. It is sure that the Cerebral ischemia reperfusion models were made successfully. Neurological deficit scores at 2h of IR and EA group were significantly higher than sham group (P<0.01). Difference of 2h、6h、7d subgroups between IR group and EA group was also significant(P<0.05).While difference at Id and 3d was obvious(P < 0.01).It prompted electric acupuncture can reduce the degree of neurological function damage in rats after cerebral ischemia reperfusion injury. Early EA intervention effects and the treatment effect is more and more obvious as time goes on. The best time to improve nerve function damage situation was the third day. Then the effect maintained stable.Cerebral infarction volume:The staining on brain tissue of sham group is normal, while IR and EA group showed different degrees of infarcts by analyzing the images and data after TTC staining. Compared with IR group, cerebral infarction volume at 6 h subgroups of EA group was reduced (P<0.05), while at 1 d,3 d,7 d subgroups cerebral infarction volume was reduced significantly(P< 0.01). Electric acupuncture treatment can reduce cerebral infarction volume.Expression of p-ERK and p-p38MAPK:The expression of p-ERK and p-p38MAPK in each subgroup of the sham group was little and the expression level showed no difference between groups (p> 0.05). Differences between IR group and sham group, as well as EA group and sham group were statistically significant (P< 0.05). Expression of p-ERK and p-p38MAPK at 2h in both groups increased. The expression of p-ERK reached the peak at 6h. P-p38MAPK expressed much in 1 d,3 d (p< 0.01). Compared with sham group, a rise appeared at 2h after CIR in EA group(p< 0.05). It increased obviously at 1d and 3d with statistically significant (p< 0.01). The expression of p-p38MAPK at each time subgroup of EA group was decreased. During the treatment, the inhibition to p-p38MAPK was obvious at 1d, 3d subgroup (P<0.01).Expression of p-JNK and p-p38MAPK:EA treatment can inhibit the expression of both p-JNK and p-p38MAPK. The expression trend of p-p38MAPK roughly as the same as before. Expression of p-JNK was visible in the sham group, but there was no statistically significant difference between different subgroups (p> 0.05). The expression of p-JNK in IR group and EA group was increased compared with the sham group. The expression of p-JNK began to decline after reach the peak at Id. Compared with IR group, the expression of p-JNK in EA group showed a drop at 2h (p< 0.05). The difference between IR group and EA group at 6h,7d was statistically significant (p< 0.05), while at 1d,3d the expression of p-JNK decreased significantly (P< 0.01).Expression of p-ERK and p-JNK:There was little expression of p-ERK and p-JNK in the sham group, and there was no significant difference in the expression level between each subgroups (P>0.05). There was significant difference between IR group, EA group and sham group (P<0.05). P-ERK of IR group started to upregulate at 2h, while reached the peak at 6h,then begained to decline. The expression of p-JNK reached the maximum at 1d. Compared with the IR group, the expression of p-ERK upregulated,while p-JNK down regulated at 2h(P< 0.05). Especially, the expression of p-ERK upregulated obviously at Id,3d subgroups, the difference between was significant (P< 0.01). The difference of p-JNK expression between IR group and EA group was statistically significant at 6h、7d (P<0.05),while decreased significantly at 1d、3d (P<0.01).ConclusionElectro acupuncture can reduce cerebral infarction volume,also alleviate nerve function damage degree caused by ischemia reperfusion. This suggests that electro acupuncture could improve brain tissue damage caused by ischemia reperfusion.The mechanisms of EA promoting the repair of damaged brain tissue, thus protecting the brain tissue maybe regulate the activation of MAPK signal pathway comprehensively. Namely, " Electro acupuncture stimulation-regulating the activation of each member of MAPK signal pathway comprehensively-antagonizing apoptosis-protecting brain function."... |
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