Recombinant tissue plasminogen activator enhances microglial cell recruitment after stroke in mice

The effect of recombinant human tissue plasminogen activator (rtPA) on neuroinflammation after stroke remains largely unknown. Here, we tested the effect of rtPA on expression of cellular adhesion molecules, chemokines, and cytokines, and compared those with levels of inflammatory cell recruitment, brain injury, and mortality over 3 days after transient middle cerebral artery occlusion (MCAO) in mice. Mortality was dramatically increased after rtPA treatment compared with saline treatment during the first day of reperfusion. Among the animals that survived, rtPA significantly increased CCL3 expression, microglia recruitment, and cerebral infarction 6 hours after MCAO. In contrast, the extent of neutrophils and macrophages infiltration in the brain was similar in both saline- and rtPA-treated animals. Recombinant human tissue plasminogen activator induced Il1b and Tnf expression, 6 and 72 hours after MCAO, respectively, and dramatically reduced interleukin 6 (IL-6) level 24 hours after reperfusion. A dose response study confirmed the effect of rtPA on CCL3 and Il1b expressions. The effect was similar at the doses of 1 and 10 mg/kg. In conclusion, we report for the first time that rtPA amplified microglia recruitment early after stroke in association with a rapid CCL3 production. This early response may take part in the higher susceptibility of rtPA-treated animals to reperfusion injury.     

褐藻多糖硫酸酯对大鼠脑缺血再灌注损伤后的神经保护作用

目的探讨褐藻多糖硫酸酯（FPS）对脑缺血再灌注损伤的神经保护作用及可能机制。方法将48只大鼠按照随机数字表法分为假手术组、大脑中动脉栓塞（MCAO）组、FPS低剂量组（MCAO＋FPS 50 mg/kg）、FPS高剂量组（MCAO＋FPS 100 mg/kg）,每组12只。假手术组大鼠只分离右侧颈总动脉及颈内外动脉,MCAO组、FPS低剂量组、FPS高剂量组采用线栓法制作大脑中动脉栓塞模型,缺血2 h后再灌注后FPS低、高剂量组予鼠尾静脉注射FPS50、100 mg/kg。对所有大鼠按照Longa标准进行神经功能评分;采用TTC法测量脑梗死体积;蛋白印迹法测定Nrf2蛋白的表达。结果与MCAO组比较,FPS低、高剂量组大鼠神经功能评分明显降低、转录因子NF-E2相关因子2（NF-E2-related factor 2,Nrf2）蛋白表达明显增高,FPS高剂量组神经功能评分、Nrf2蛋白表达改变得更为显著（P〈0.05,P〈0.01）;FPS高剂量组大鼠脑梗死体积明显减小（P〈0.01）。结论 FPS通过提高Nrf2的表达缩小脑梗死体积,对脑缺血再灌注损伤大鼠起到神经保护作用。     

Notch信号通路对脑缺血大鼠神经干细胞移植后神经再生的影响

目的 探讨抑制Notch信号通路促进脑缺血大鼠神经干细胞（NSCs）移植后神经再生的机制。方法 采用大脑中动脉阻塞MCAO）方法构建局灶性脑缺血模型,体外培养NSCs,移植入纹状体缺血区。将40只SD大鼠随机分为假手术组、模型组、移植组（移植神经干细胞）、氮-［氮-(3,5-二氟苯乙酰)-L-丙氨酰］-S-苯基甘氨酸丁酯］（DAPT）+移植组。采用HE染色观察各组大鼠神经元损伤程度,利用免疫组织化学、Western blotting检测各组大鼠脑组织中Notch1、Hes1及Hes5的表达情况。结果 与假手术组相比,模型组神经元损伤严重,出现核固缩和核溶解,Notch1、Hes1及Hes5阳性细胞表达明显增多,Notch1、Hes1及Hes5蛋白表达显著上调（P＜0.05）。与模型组相比,移植组和DAPT+移植组神经元损伤均不同程度缓解,Notch1、Hes1及Hes5阳性细胞均有部分表达,各项蛋白表达均有所降低（P＜0.05）;DAPT+移植组神经元损伤明显恢复,较移植组各项蛋白阳性细胞和蛋白表达进一步降低(P＜0.05)。结论 抑制Notch信号通路可促进脑缺血大鼠神经干细胞移植后的神经再生,其机制主要与下调Notch1、Hes1及Hes5的表达有关。     

早期运动干预对脑梗死大鼠皮质脊髓束的影响

目的 观察早期运动干预对脑梗死大鼠皮质脊髓束的影响。 方法 取雄性SD大鼠18只,按随机数字表法分为静息组、造模1 d后运动组（1D组）、造模1周后运动组（1W组）,每组6只大鼠,3组大鼠均采用改良的Longa线栓法制备大脑中动脉闭塞（MCAO）模型。1D组大鼠在造模1 d后开始运动训练,1W组大鼠在造模1周后开始运动训练,静息组大鼠则每天被置于静止的跑台静息30 min。3组大鼠均于造模1、4、8周后,采用改良神经功能缺损评分法（mNSS）评价的神经功能,核磁共振T2WI序列计算脑梗死体积比,扩散张量成像（DTI）序列检测双侧皮质脊髓束的各向异性分数比值(rFA),并通过扩散张量纤维束成像（DTT）技术观察皮质脊髓束形态,最后分析rFA值与mNSS评分的相关性。 结果 造模1周后,1D组的mNSS评分显著低于1W组和静息组（P<0.05）;造模4周后,1D组和1W组mNSS评分均显著低于静息组（P<0.05）。造模8周后,1D组mNSS评分显著低于1W组和静息组（P<0.05）,且1W组mNSS评分也显著低于静息组（P<0.05）。造模1周和4周后,1D组的梗死体积比显著低于1W组和静息组（P<0.05）。造模4周后, 1W组梗死体积比显著低于静息组（P<0.05）。造模8周后,1D组和1W组的梗死体积比显著低于静息组的0.19±0.03（P<0.05）。造模1周后,1D组的rFA值显著低于静息组（P<0.05）。造模4周后,1D组和1W组的rFA值均显著高于静息组（P<0.05）。造模8周后,1D组rFA值显著高于1W组和静息组,同时1W组的rFA值高于静息的0.49±0.09,差异均有统计学意义（P<0.05）。DTT显示,造模8周后,1D组大鼠的皮质脊髓束比1W组和静息组对称性更好。相关性分析显示,rFA与mNSS评分有较高的相关性（r=-0.707,P=0.001）。 结论 运动干预可以促进脑梗死大鼠皮质脊髓束重塑,改善神经功能,造模1 d后即开始早期运动效果更佳。     

Loss of plasma membrane integrity,complement response and formation of reactive oxygen species during early myocardial ischemia/reperfusion

Loss of plasma membrane integrity (LPMI) is a hallmark of necrotic cell death. The involvement of complement and ROS in the development of LPMI during the early stages of murine myocardial ischemia–reperfusion injury was investigated. LPMI developed within 1 h of reperfusion to a level that was sustained through 24 h. C3 deposition became significant at 3-h reperfusion and thus contributed little to LPMI prior to this time. SOD1 transgenic mice had significantly less LPMI compared with WT mice at 1 h of reperfusion but not at later time points. Catalase transgenic mice were not protected from LPMI at 1-h reperfusion compared with WT mice, but had 69% less LPMI at 3-h reperfusion. This protection was transient. At 24-h reperfusion the LPMI of catalase transgenic mice was identical to that of WT mice. The delayed benefits of over-expressed catalase compared with SOD1 are consistent with its antioxidant action downstream of SOD1. The onset of LPMI occurs within 1 h of reperfusion at a level that is maintained through 24 h. ROS contribute significantly to LPMI during the first 3 h of reperfusion, while complement deposition, which becomes significant after 3-h reperfusion, may contribute thereafter.     

Early detection of abnormal left ventricular relaxation in acute myocardial ischemia with a quadratic model

AimsThe time constant of left ventricular (LV) relaxation derived from a monoexponential model is widely used as an index of LV relaxation rate, although this model does not reflect the non-uniformity of ventricular relaxation. This study investigates whether the relaxation curve can be better fitted with a “quadratic” model than with the “conventional” monoexponential model and if changes in the LV relaxation waveform due to acute myocardial ischemia could be better detected with the quadratic model.Methods and resultsIsovolumic relaxation was assessed with quadratic and conventional models during acute myocardial ischemia performed in 6 anesthetized pigs. Mathematical development indicates that one parameter (Tq) of the quadratic model reflects the rate of LV relaxation, while the second parameter (K) modifies the shape of the relaxation curve. Analysis of experimental data obtained in anesthetized pigs showed that the shape of LV relaxation consistently deviates from the conventional monoexponential decay. During the early phase of acute myocardial ischemia, the rate and non-uniformity of LV relaxation, assessed with the quadratic function, were significantly enhanced. Tq increased by 16% (p < 0.001) and K increased by 12% (p < 0.001) within 30 and 60 min, respectively, after left anterior descending (LAD) coronary artery occlusion. However, no significant changes were observed with the conventional monoexponential decay within 60 min of ischemia.ConclusionsThe quadratic model better fits LV isovolumic relaxation than the monoexponential model and can detect early changes in relaxation due to acute myocardial ischemia that are not detectable with conventional methods.     

Lysophosphatidic Acid Rescues Human Dental Pulp Cells from Ischemia-induced Apoptosis

Introduction

Dental pulp is particularly susceptible to ischemic conditions (hypoxia and serum deprived) because it is commonly exposed to trauma, inflammation, chronic caries injury, and pulpitis. We investigated the apoptotic response of human dental pulp cells (HDPCs) to varying levels of oxygen and serum to mimic different degrees of ischemia, tested whether lysophosphatidic acid (LPA) could reverse ischemia-induced apoptosis, and investigated the possible mechanisms of LPA.

Methods

HDPCs were cultured under conditions mimicking serum deprivation and ischemia for 2 days with or without LPA at 25 μg/mL. Flow cytometry and JC-1 fluorescence were used to detect any apoptotic change. Western blotting was used to measure the expression of the apoptosis regulators B-cell lymphoma 2 (Bcl-2) and Bax, focal adhesion kinase (FAK), Src, extracellular signal-regulated kinase (ERK), and Akt.

Results

Flow cytometry and JC-1 immunofluorescence showed that ischemia could induce apoptosis of HDPCs in 2 days and treatment with LPA could reduce cell death significantly. To clarify the molecular mechanisms, Western blot results showed up-regulation of both proapoptotic Bax and antiapoptotic Bcl-2 during apoptosis. LPA functioned as an antiapoptotic cytokine by activation of the phosphorylation of FAK and ERK. No statistically significant difference was found in the activation levels of p-Src or p-Akt.

Conclusions

A self-defense mechanism functioned during cell apoptosis. LPA could effectively rescue HDPCs from ischemia-induced apoptosis via regulation of Bax and Bcl-2 and the activation of phosphorylated FAK and phosphorylated ERK. LPA is a potent candidate for biological therapy of chronic pulpal inflammatory diseases.     

Influence of ezetimibe on selected parameters of oxidative stress in rat liver subjected to ischemia/reperfusion

Introduction

Ischemia/reperfusion (I/R) is considered to be one of the main causes of liver damage after transplantation. The authors evaluated the effect of ezetimibe on selected oxidative stress parameters in ischemic/reperfused (I/R) rat liver.

Material and methods

Rats were administered ezetimibe (5 mg/kg) (groups E and E-I/R) or saline solution (groups C and C-I/R) intragastrically for 21 days. Livers of animals in groups C-I/R and E-I/R were subjected to 60 min of partial ischemia (left lateral and median lobes) followed by 4 h of reperfusion. Alanine and asparagine aminotransferase (ALT, AST) activity was determined in blood before I/R and during reperfusion (at 15 and 240 min). After the reperfusion period, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and glutathione peroxidase (GPx) were determined in liver homogenates using colorimetric methods.

Results

Ezetimibe caused a significant increase in GSH level in groups subjected to I/R (E-I/R (99.91 ±9.01) vs. C-I/R (90.51 ±8.87), p < 0.05). Additionally, under I/R the decrease of GPx activity in the drug-treated group was lower compared to the non-treated group (E-I/R (3.88 ±1.11) vs. E (5.31 ±1.83), p = 0.076). Neither ezetimibe nor I/R affected SOD or MDA levels. I/R produced a significant increase in aminotransferase levels (ALT240-0: C-I/R (42.23 ±43.56) vs. C (9.75 ±11.09), and E-I/R (39.85 ±26.53) vs. E (4.38 ±1.36), p < 0.05 in both cases; AST 240-0: E-I/R (53.87 ±17.23) vs. E (24.10 ±9.66), p < 0.05) but no effect of ezetimibe on those enzymes was found.

Conclusions

Ezetimibe demonstrates antioxidant properties in rat livers subjected to I/R. However, neither a hepatoprotective nor a hepatotoxic effect of ezetimibe was demonstrated, regardless of I/R.