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OBJECTIVE：To study the improvement effects of icariside Ⅱ（ICS Ⅱ）on neurological function of focal cerebral ischemia model rats by regulating miR- 141-3p/Notch/nuclear factor erythroid- 2-related factor 2（Nrf2）axis（miR-141-3p/Notch/ Nrf2）. METHODS ：The rats were divided into sham operation group ，model group ，nimodipine group （20 mg/kg）and ICS Ⅱ low-dose，medium-dose and high-dose groups （4，8，16 mg/kg），with 20 rats in each group. Twenty-four hours after establishing focal cerebral ischemia model ，model rats were given re levant medicine or normal saline intragastrically ，twice a day ，for consecutive 3 d. The neurological deficit of rats in each group was scored ；the volume of cerebral infarction was measured by 2，3， 5-triphenyltetrazolium chloride （TTC）staining；water content of cerebral tissue and the permeability of blood-brain barrier were measured；HE staining was performed to observe the pathological change of cerebral tissue of rats ；the expression of miR- 141-3p in cerebral tissue of rats was measured by qRT-PCR ；the protein expression of Notch and Nrf 2 in cerebral tissue of rats were measured by Western blotting assay. RESULTS ：Compared with sham operation group ，the neurological deficit score ，expression of Notch-1 and Nrf 2 in model group were significantly lowered （P＜0.05）；infarction volume ，brain water content ，the permeability of blood-brain barrier and the expression of miR- 141-3p in cerebral tissue were increased significantly （P＜0.05）；the distribution of cortical cells was disordered ，and inflammatory infiltration and necrosis were observed in a large number of nerve cells. Compared with model group ，the neurological deficit score ，the protein expression of Notch- 1 and Nrf 2 in cerebral tissue were significantly increased in ICS Ⅱgroups（P＜0.05）；infarction volume ，brain water content ，the permeability of blood-brain barrier and the expression of miR- 141-3p in cerebral tissue were decreased significantly （P＜0.05）；the arrangement of cortical cells was regular，and the inflammatory infiltration and necrosis of nerve cells were decreased significantly. CONCLUSIONS ：ICS Ⅱ can promote the recovery of neurological function in focal cerebral ischemic model rats ，which may be related to down-regulation of miR-141-3p and activation of Notch/Nrf 2 axis.
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Objective: To establish a UPLC-MS/MS analysis method for determination of baicalin, geniposide, chlorogenic acid, cholic acid and hyodeoxycholic acid in Qingkailing (lyophilized) for injection in rat plasma, and to investigate the pharmacokinetic behavior of this preparation in normal and cerebral ischemic rats. Method: Rats were randomly divided into normal group and cerebral ischemia model group. The rat model of cerebral ischemia was established by suture embolization. The rats were given by intraperitoneal injection, and normal saline was used as the solvent. Blood samples were taken at the corresponding time points. After treatment, UPLC-MS/MS was used to determine the blood concentration of five components. The main detection conditions were mobile phase of 0.1%formic acid aqueous solution-acetonitrile for gradient elution (0-0.25 min, 90%A; 0.25-1 min, 90%-75%A; 1-2 min, 75%-50%A; 2-2.6 min, 50%-45%A; 2.6-2.65 min, 45%-90%A; 2.65-4.0 min, 90%A), the flow rate of 0.4 mL·min-1, the column temperature at 40℃, electrospray ionization under negative ion mode. The pharmacokinetic parameters were fitted and the bioavailability was calculated, the differences of treatment process of five components from Qingkailing (lyophilized) for injection in normal and cerebral ischemic rats were analyzed. Result: Compared with the normal group, the area under the curve (AUC0-t) of geniposide in rats from cerebral ischemia model group decreased significantly after intraperitoneal injection of Qingkailing (lyophilized) for injection (PTmax) of chlorogenic acid in rats from cerebral ischemia model group was significantly earlier than that in the normal group (PConclusion: Qingkailing (lyophilized) for injection has a certain difference in the treatment process between normal and cerebral ischemic rats, which has certain guiding significance for the clinical treatment of cerebral ischemic diseases with this preparation.
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Objective To investigate the establishment of focal cerebral ischemia model in rabbits with the improved suture method.Methods A total of 45 healthy and clean adult New Zealand rabbits were divided into either a sham operation group (n =5) or a model group (n =40) using random number table method before modeling,and the sex was not limited.The self-made head ends of 2-0 fishing lines dipped in paraffin were used as the sutures.The external carotid artery was cut and inserted into a intracranial artery through the internal carotid artery and blocked the origin of middle cerebral artery.The neurological function score was performed after 6 h.The neurological deficit scores ≥2 was successful modeling.The rabbits were killed by anesthesia.The brain slices were stained with 2％ 2,3,5-triphenyl tetrazolium chloride solution.The infarct foci were observed.The diameters of suture head and the depth of suture insertion were compared in the model rabbits with successful modeling,failure,and death in the model group.Results There were 40 rabbits in the model group,six of them died,including 4 died of subarachnoid hemorrhage within 4 h after operation,and 2 died from anesthetic accident.The mortality rate was 15.0％.Seven rabbits failed,mainly because of cerebral vasospasm and the insertion depth of suture was insufficient.Twenty-seven had successful modeling,and the success rate was 67.5％.All the rabbits in the sham operation group survived.The diameter of the suture head and insertion depth in the successful modeling rabbits were compared with the death and failure outcome in rabbits.The difference was statistically significant (diameter:0.52 ± 0.14 mm vs.0.45 ±0.40 mm and 0.58 ±0.17 mm;depth:5.49 ±0.17 cm vs.6.04 ± 0.11 cm and 4.26 ±0.30 cm;all P ＜ 0.05).Conclusions The improved suture method can successfully prepare the focal cerebral ischemia model of middle cerebral artery in rabbits.The method is simple.Its repeatability and practicability are better.
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@#There are many ways to prepare middle cerebral artery occlusion (MCAO) rat model. The focal cerebral ischemia model es-tablished by the suture-occluded method is widely accepted as an ideal focal cerebral ischemia model, which can be used in the basic and clinical study of cerebral vascular diseases. However, it still has the defects of high technical requirement, large model difference and high animal mortality. A variety of focal cerebral ischemia models were established by changing the retention time of the thread, the road the thread plugging into, the type of thread bolt and the depth of the thread plugging. This article compared different suture-occluded method, as well as the suture-occluded method to other modeling methods, different cerebral ischemia models may be different in the pathophysiology of cerebral infarction, and the effects may also be different.
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Middle cerebral artery occlusion model with suture-occluded method in rats is widely regarded as the standard animal model of focal cerebral ischemia .In the process of preparation , it is easy to be influenced by multiple factors;among them the successful operation plays a key role .This article summarized from several aspects , such as the anatomic locations of the major arteries in the rat ’ s neck , where to perform the incision , which side to perform the operation and where to insert the suture .
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@#Objective To compare and evaluate the efficacy of three operating procedures to produce permanent rat models of cerebral ischemia, including mortality, neurological evaluation score, infarction volume of ischemia and time consumed in the operation. Methods The rats were randomly divided into three groups. Group 1: The common carotid artery (CCA) and external carotid artery (ECA) were ligated during the operation and ICA was clipped temporarily by artery clamp. Group 2: The CCA and ECA were ligated and the superior thyroid artery and pterygopalatine artery were exposed but not ligated. Silk suture was utilized to hang the internal carotid artery (ICA). Plastic suture was inserted into ICA and stepped over the initiation point of pterygopalatine artery under microscopic observation. Group 3: The CCA and ECA were ligated and ICA was hanged with a silk suture, and the pterygopalatine artery was not exposed, but during the period of inserting plastic suture, the proximal part of the suture was pressed to make the suture's round distal end elevated, and then pass the initial point of pterygopalatine artery. Mortality, neurological score, volume of ischemic infarction and operation time consumed of the three groups were compared after the operation. Results The procedure to prepare the model was most efficient in the group 3, taking only 17.5 min to complete, significantly less than that in the group 1 (50 min) and group 2 (40 min), (P<0.05), and with a lower mortality and more steady neurological evaluation score and infarction volume. Conclusion The use of the third operating method can shorten the operation time and improve the efficacy of operation. Using this method, more consistent and repetitive focal cerebral ischemia models can be produced effectively, and meet the demands of clinical trials.
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Middle cerebral artery occlusion(MCAO) can be used to establish the standard model of focal cerebral ischemia.The method for establishing focal cerebral ischemia model has been improved continuously,from simple behavior score,measurement of infarcted brain area,and morphological observation in the past to functional study,neurobiochemistry,molecular biology,etc.The research of MCAO provides a basis for further study of the pathological characteristics,mechanism and prevention of human cerebral ischemia.This article reviews the recent advancement in the study of MCAO model and cerebral(ischemia.)
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Objective To study the role of caspase 3 in ischemic brain damage of rats, and further understand the molecular mechanisms of ischemic cerebral vascular disease.Methods Rat models of the left middle cerebral artery (MCA) occlusion/reperfusion were made using a modification of the intraluminal sature method of Longa established by Belayev, infarct zones were confirmed by 2,3,5 triphenyltetrazolium chloride (TTC) staining, and caspase 3 expression on brain sections at the mRNA and active protein level was detected with in situ hybridization and immunohistochemistry technique, respectively.Results After 2 hours of left MCA ischemia followed by 24 hours of reperfusion, obvious infarct in the MCA dominate regions was confirmed by TTC staining; low levels of caspase 3 mRNA, and fewer of its active protein expression was found in normal brains, sham brains and contralateral brains of MCAO rats; both caspase 3 mRNA and activated protein expression in ipsilateral region were increased after 24 hours of recirculation, and even higher levels were detected at 48 hours of reperfusion.Conclusion Apoptotic mechanism might involve in delayed neuronal death after cerebral ischemia, and caspase 3 might play an important role in ischemic neuronal injury.