Protective Effects of Sesamol on the Brains of Rats with Focal Cerebral Ischemia-Reperfusion Injury

Research Article

Austin J Pathol Lab Med. 2021; 8(1): 1032.

Protective Effects of Sesamol on the Brains of Rats with Focal Cerebral Ischemia-Reperfusion Injury

Guannan X, Xiujuan G, Xin L, Yujue Z, Zhaoyong L, Ximeng C* and Fengzhen L*

Liaocheng People’s Hospital, Medical College of Liaocheng University, Liaocheng Dongchangfu Hospital, Liaocheng, P. R. China

*Corresponding author: Chen Ximeng, Liaocheng People’s Hospital, Medical College of Liaocheng University, Liaocheng Dongchangfu Hospital, Liaocheng, 252000, P. R. China

Liu Fengzhen, Liaocheng People’s Hospital, Medical College of Liaocheng University, Liaocheng Dongchangfu Hospital, Liaocheng, 252000, P. R. China

Received: April 16, 2021; Accepted: May 01, 2021; Published: May 08, 2021


Stroke is an acute cerebrovascular event associated with brain tissue injury, representing the most common cause of death. Thrombolysis and recanalization are the principal treatment modalities for ischemic stroke. Some patients experience cerebral ischemia-reperfusion injury following treatment. A previous study established that sesamol is effective in reducing risk factors for stroke. Here, we aimed to investigate the protective effect of sesame phenol on cerebral ischemia-reperfusion injury. A total of 72 SD rats were randomly divided into a sham group, cerebral ischemia-reperfusion (MCAO) group, cerebral ischemia-reperfusion with low dose sesame phenol (MCAO+sesamol A) group and cerebral ischemia-reperfusion with high dose sesame phenol (MCAO + sesamol B) group. After cerebral ischemia had been induced for 2h and reperfusion conducted for 24h, the volume of cerebral infarction, the degree of cerebral edema and the neurological deficit scores were tested. The results showed sesamin improved the neurological deficit score in a dose-dependent manner, reduced the volume of cerebral infarction, degree of cerebral edema. Prophylactic treatment with sesame phenol provided neuroprotective effects on cerebral ischemia-reperfusion injury.

Keywords: Cerebral ischemia-reperfusion injury; Sesamol; Neuroprotective effect; Cell apoptosis


Stroke is an acute cerebrovascular event associated with brain tissue injury. Stroke represents the most common cause of severe permanent disability and the second most common cause of death and dementia. Thrombolysis and recanalization are the principal treatments for ischemic stroke. However, the time window for treatment of recombinant Plasminogen Activator (rtPA) thrombolysis is narrow [1], and requirements for its application strict. Due to specific indications and contraindications, only 2% to 5% of stroke patients have the opportunity to receive this form of treatment, even if the probability of success in patients with Cerebral Ischemia Reperfusion Injury (CIRI) is up to 50% [2]. The pathophysiological mechanisms that result in CIRI are complex, with multiple lesion cascades involved that form a complex network of pathological mechanisms. A number of anti-inflammatory agents and antioxidants have demonstrated particular therapeutic effects in animal experiments, but the results have not been replicated in multiple clinical trials. An important reason is that the mechanisms of action of these drugs encompasses only a fraction of the many mechanisms involved in stroke, rather than representing a multitarget, comprehensive treatment for disease networks. In addition, a number of drugs cannot provide the expected clinical effect due to particular toxic side effects. Therefore, in recent years, the search for a natural plant with active ingredients having multiple biological activities has been an intense focus of research interest, in order to achieve a multi-target and multi-channel treatment for stroke without toxic side effects, so as to provide a broader range of possibilities for the development of novel neuroprotective agents.

Sesamol (also known as 5-hydroxy-1, 3-benzodioxole), a natural extract of flax, is an aromatic component of sesame oil and a principal degreased antioxidant. After passing through the blood-brain barrier, it exhibits a variety of biological properties, being anti-inflammatory, an anti-oxidant, antihypertensive, and reducing apoptosis and lowering lipids [3]. It has recently been demonstrated that sesame phenol has neuroprotective properties, having the capability to maintain the integrity of cells in the neurovascular unit, protect the blood-brain barrier [4], and enhance cognitive function [5]. However, the effects of sesame phenol on acute cerebral ischemia-reperfusion injury have rarely been reported. If would be of considerable clinical significance if sesame phenol were demonstrated to have neuroprotective properties with multiple targets and dimensions. In this study, SD rats in which occlusion of the middle cerebral artery were used to investigate the effects of sesame phenol on CIRI.

Materials and Methods


Experimental animals and groups: Male SD rats (body weight: 170-200 g) that were specific-pathogen free (SPF) were purchased from Shandong Jinfeng Experimental Animal Co., Ltd. (License No.: SCXK (Lu) 2014 0006). The procedures involving animals and their care were approved by the Animal Care and Use Committee of Liaocheng People’s Hospital (Grant No.: LY20160916). The SD rats were randomly allocated into one of 4 equal groups according to a random number table method: sham surgery (sham) group, focal middle cerebral artery occlusion (MCAO) group; MCAO+sesamol group A and MCAO+sesamol group B, the latter two receiving MCAO surgery, but with sesame phenol (98% purity) at doses of 5mg·kg–1·d–1 and 25mg·kg–1·d–1, injected intraperitoneally for 7 days prior to MCAO. The sham and MCAO groups were injected intraperitoneally continuously in the same manner but with physiological saline as control treatments.

Principal reagents and equipment: Sesamin (BSZH–Z–021, Sigma–Aldrich Co. Ltd), TTC solution (T8877–5G, Sigma– Aldrich Co. Ltd), DAB chromogenic kit (Nanjing Institute of Bioengineering), TUNEL staining kits, Caspase–3 antibody kits and SABC immunohistochemical staining kits were purchased from Wuhan Bude Biotechnology Engineering Co., Ltd. The following reagents and equipment were sourced as follows: goat anti–rabbit IgG/horseradish peroxidase (ZB2303, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), laser doppler blood flow monitor (PeriFlux, Stockholm, Sweden), paraffin slicer (Shandon 325, Shandon, UK), embedding box (Jiangsu Shitai Experimental Equipment Co., Ltd.), digital camera (Olympus, Japan) and image analysis system (Image– Pro Plus 6.0).


Establishing the MCAO model: The MCAO model consisted of the wire plug method described by Chen [6]. The wire plug consisted of a 0.15mm diameter, 4cm long sterile nylon filament thread (20mm of one end coated with polylysine) and the other end coated with silica gel. Rats were anesthetized with 3% isoflurane and maintained with 2% isoflurane in a gas mixture of 5% CO2 and 95% O2. During the entire procedure, temperature was maintained at 370C using a thermostatically controlled heated blanket. For the MCAO model, blood flow in the obstructed artery was monitored using a doppler blood flow monitor in real time. When blood flow had decreased by more than 85%, the experiment was deemed to have begun. At this stage, the color of the iris became lighter, then white, and the skin was sutured layer by layer, with no active bleeding observed. The nylon filament was tightened for 2h, and then reperfusion allowed for 24h. The middle cerebral artery in the rats in the sham group was exposed but not subjected to ischemia–reperfusion surgery. During surgery, if a rat developed dyspnea or excessive bleeding, there were replaced by an additional rat, such that the number of animals in each group was 18. The vital signs of the rats were monitored during the perioperative period, and HR, RR, SPO2 and T recorded 5min prior to MCAO, 5min afterward, 5min before reperfusion, and 5min after reperfusion.

Evidence that MCAO was successfully performed included development of Horner syndrome on the same side as ischemia, with hemiplegia of the contralateral forelimb following recovery from anesthesia.

Neurological deficit score: After 2h of cerebral ischemia and 24h of reperfusion, the neurological deficit of the rats using criteria from the Christa report [7] were recorded, in a blinded–fashion. The score comprises 6 tests: 1) Autonomic movement of the rats; 2) Symmetry of limb movement; 3) Forelimb extension function; 4) Climbing movement; 5) Proprioceptive function; Each test was scored from 0 to 3, with a minimum neurological score of 0 and a maximum of 18 points. Lower scores represented more severe neurological dysfunction.

Measurement of volume of cerebral infarction: Six rats in each group were randomly injected with 3% isoflurane and maintained with 2% isoflurane in a gas mixture of 5% CO2 and 95% O2 for anesthesia. The rat was quickly decapitated then the head placed on ice, after which blood was washed away and the head frozen at –20oC in a freezer for 30 min. The volume percentage of cerebral infarction was calculated according to the method described by Qiu ZD: the percentage of cerebral infarction volume (%) = [total cerebral infarction volume – (volume of right brain hemisphere – volume of left hemisphere)] / total volume of left brain tissue × 100% [8].

Calculation of water content in ischemic brain tissue: After cerebral ischemia for 2h and reperfusion for 24h, 6 rats in each group were randomly selected for decapitation, and the water content of the brain tissue measured using the dry and wet weighing method. The weight was immediately measured, representing the wet weight (G1); brain tissue was wrapped in aluminum foil, baked within a constant temperature drying oven at 1100C until the weight had equilibrated, representing the dry weight (G2). Brain tissue water content = [(G1– G2) / G1] × 100%.

Statistical methods: SPSS v16.0 software was used to conduct statistical analysis on the data. A Kolmogorov–Smirnov analysis was used for the testing of normality, the Levene method for assessing homogeneity of variance, and a comparison of multiple sets of data performed by one–way ANOVA. Multiple comparisons of sample means were evaluated using a Tukey test, and a nonparametric rank sum test used when there was no difference in variance. The vital signs of the rats were analyzed by repeated measures of variance, and the Bonferroni method used for comparisons between groups. The test level was a=0.05, with P<0.05 considered statistically significant. Graph pad Prime 5.0 software was used to plot graphs.


Comparison of general information

Modeling: In the present study, 82 SD rats were used, of which 8 rats were excluded from the cerebral ischemia–reperfusion injury model. Of these, 5 died due to subarachnoid hemorrhage, 2 suffered severe cerebral edema and respiratory depression occurred in 1 when the vagus nerve was isolated. No deaths occurred in the SD rats in the sham group. The success rate of the modeling was 84%, with mean duration of 22min.

Regional cerebral blood flow in each group: Before MCAO, there was no significant difference in the regional Cerebral Blood Flow (rCBF) compared with 5min after MCAO, 5min before reperfusion, and 5min after reperfusion (Figure 1).