Early increase in mRNA levels of pro-inflammatory cytokines and their interactions in the mouse hippocampus after transient global ischemia

There is convincing evidence that cytokines are involved in the inflammatory response following cerebral ischemia, but the interactions among the pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6 in the early stage of ischemic reperfusion are not yet completely understood. In this study, we examined the early mRNA expressions of pro-inflammatory cytokines in the ischemic hippocampus after 30 min of bilateral common carotid artery occlusion in C57BL/6J wild-type (WT) and TNF-alpha, IL-1alpha/beta or IL-6 gene knockout (KO) mice utilizing real-time polymerase chain reaction. The mRNA expressions of the pro-inflammatory cytokines TNF-alpha, IL-6 and IL-1beta were significantly induced in ischemic WT mice compared with in the sham-operated mice. These increases peaked at 3 to 24 h for TNF-alpha, at 12 h for IL-1beta, and at 6 to 24 h for IL-6 after ischemia. The pattern of temporal expression of the cytokine mRNAs in ischemic gene KO mice, however, differed from that in WT mice. The TNF-alpha mRNA expression showed a similar temporal expression pattern in IL-6 KO mice compared to in WT mice following ischemic reperfusion, and the levels at all time points were lower than in WT mice. The IL-1beta mRNA level was very low in ischemic TNF-alpha KO mice and IL-6 KO mice in spite of a small peak observed in both at 24 h. The IL-6 mRNA level was significantly upregulated at all time points in both ischemic WT and TNF-alpha KO mice; however, the peak was delayed by 12-h in IL-1alpha/beta KO mice. In conclusion, the present study indicates that the rapid increases in cytokine levels are interdependent, interactive, and possibly modulate each other in the mouse hippocampus after transient global ischemia.     

Permanent occlusion of the middle cerebral artery upregulates expression of cytokines and neuronal nitric oxide synthase in the spinal cord and urinary bladder in the adult rat

The expression pattern of proinflammatory cytokines, neuronal nitric oxide synthase (nNOS), substance P (SP) and calcitonin gene related peptide (CGRP) in the spinal cord and the bladder in response to permanent middle cerebral artery occlusion (MCAO) was investigated. In this connection, the gene expression of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta) and interleukin-6 in the lumbosacral spinal cord and the bladder as determined by real-time polymerase chain reaction was upregulated. In the spinal cord, the immunoreactivity of TNF-alpha and IL-1beta was mainly localized in the ventral horn motoneurons contralateral to MCAO. In the bladder, TNF-alpha was mainly expressed in the inflammatory cells. The expression of nNOS immunoreactivity as well as nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining in the spinal cord and bladder was also markedly increased in response to MCAO. Furthermore, the temporal and spatial expression of nNOS paralleled that of TNF-alpha and IL-1beta in the spinal cord. On the other hand, there was no noticeable change in gene expression and immunoreactivity of SP and CGRP. The present results have shown that cytokines and nNOS expression are elevated in areas far removed from the primary site of ischemic infarct, namely, the lumbosacral spinal cord and bladder. This together with some neuronal deaths maybe linked to the dysfunction of the latter in a clinical stroke. On the other hand, the apparent lack of SP and CGRP changes following MCAO suggests that the two neurotransmitters are not directly involved.     

Delayed treatment of Na+/H+ exchange inhibitor SM-20220 reduces infarct size in both transient and permanent middle cerebral artery occlusion in rats

SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulfonate) is a Na+/H+ exchanger (NHE) inhibitor which has been shown to attenuate cerebral edema in the rat transient focal ischemia model. However, to date, the effect of SM-20220 on cerebral infarction has not been examined. The present experiments were designed to investigate these effects, using both transient and permanent middle cerebral artery (MCA) occlusion models in rats. A dose of 1 mg/kg given intravenously 30 min after the onset of transient MCA occlusion reduced the infarcted area. In the permanent MCA occlusion model, SM-20220 reduced the infarcted area when treatment was delayed for 5, 30 or 60 min after the onset of ischemia. The present results show that NHE has a crucial role in the pathogenesis of ischemic brain damage. This NHE inhibitor may be useful for treating stroke because of its effectiveness with both forms of ischemia and because of its postischemic administration.     

Possible involvement of NO/NOS signaling in hippocampal amyloid-β production induced by transient focal cerebral ischemia in aged rats

In the present study, to define the roles of nitric oxide (NO) signaling in amyloid-β (Aβ) production after transient cerebral ischemia, extracellular levels of NO and Aβ were monitored by intracerebral microdialysis in the hippocampus of aged rats exposed to transient middle cerebral artery occlusion and reperfusion (MCAO/R). The results indicated that 1-h MCAO significantly upregulated hippocampal NO and Aβ levels. In addition, the NO elevation preceded the Aβ changes. The Western blotting suggested that acute hypoperfusion could increase the expression of β-secretase 1 (BACE1) but not BACE2. The enhanced NO concentration in acute stage of MCAO/R was coincident with increased eNOS expression, while in subacute stage was coincident with increased iNOS and nNOS. Our results also indicated that pretreatment of L-NAME, one non-selective NOS inhibitor could decrease the BACE1 expression, reverse both NO and Aβ changes and rescue the delayed neuronal death. These preliminary findings indicated that activation of NOS/NO signaling system could trigger Aβ production through BACE1 pathway during acute ischemic episode. The present data may be important in understanding, at least in part, the pathological role of NO/NOS system involved in hippocampal Aβ production and neuronal damage induced by transient cerebral ischemia.     

辣椒素受体拮抗剂AMG517在小鼠脑缺血/再灌注损伤中通过调节炎症因子释放发挥神经保护作用#br#

目的 观察辣椒素受体(TRPV1)拮抗剂AMG517在小鼠脑缺血/再灌注损伤中的作用及相关机制。方法 40只雄性C57BL/6小鼠随机分为假手术组(sham,n=10)、赋形剂（vehicle）+缺血/再注灌注组(vehicle,n=10)、辣椒素+缺血/再注灌注组(capsaicin,n=10)和AMG517+缺血/再注灌组(AMG517,n=10)。采用大脑中动脉闭塞(MCAO)诱导缺血/再灌注损伤,再灌注72 h进行神经行为学评分;同时检测各组小鼠梗死体积、脑水肿、TRPV1 mRNA表达和血清肿瘤坏死因子-α(TNF-α)浓度及白细胞介素-10(IL-10)浓度。结果 与vehicle组相比,AMG517显著减小鼠脑梗死体积(P<0.01)。AMG517给药后也可显著降低小鼠神经行为学评分(P<0.01)。与sham组比较,vehicle组TRPV1 mRNA表达显著增加(P<0.01)。AMG517给药后可显著增加抗炎性细胞因子IL-10浓度,并降低炎性细胞因子TNF-α浓度(P<0.05)。结论 AMG517可改善小鼠缺血/再灌注损伤,可能是通过缓解炎症发挥神经保护作用。     

Effect of fentanyl on TNF-alpha and IL-1beta levels during global ischemia/reperfusion in rats

To reduce surgical stress, fentanyl is frequently used for neurosurgical procedures in which focal and/or global ischemia may occur. However, the effect of fentanyl on cytokine levels during ischemia/reperfusion is still uncertain. The goal of this study was to evaluate the effect of fentanyl infusion on levels of the proinflammatory cytokines, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, during global cerebral ischemia/reperfusion in rats using the intracerebral microdialysis technique. Forty male Sprague-Dawley rats weighing 280-320 g were randomly assigned to each of four groups: group 1 (no fentanyl infusion and only ischemia/reperfusion); group 2 (1.5 ng/ml of fentanyl infusion during ischemia/reperfusion) and group 3 (3 ng/ml of fentanyl infusion during ischemia/reperfusion) (n=5 in each group). The rats were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg). They were then intubated and ventilated with room air using an animal ventilator. A CMA-12 probe was inserted into the left hippocampal CA-1 region according to the guidelines. Artificial cerebrospinal fluid was run from the inserted microdialysis probe and infused with or without fentanyl at 3 microl/min using a microinjection syringe pump during ischemia/reperfusion. Ischemia was induced by clamping the carotid arteries. Hemorrhagic hypotension was induced for 17 min via the femoral artery, and reperfusion was accomplished by unclamping the sling and reinfusing the blood via the femoral artery. After 2 h of stabilization, the microdialysate was collected 10 times every 17 min, just before ischemia (control), after ischemia (I) and after reperfusion (R1-R8), and stored at -80 degrees C until analysis using high-performance liquid chromatography During global ischemia/reperfusion, TNF-alpha and IL-1beta significantly increased at reperfusion (R5) compared with the control value (p < 0.05). However, in both cases of fentanyl infusion, TNF-alpha and IL-1beta showed no increase compared with the control value. Fentanyl inhibited an increase of the proinflammatory cytokines, TNF-alpha and IL-1beta levels, during global cerebral ischemia/reperfusion in rats.     

Expressions of TNF-alpha and IL-1beta in human ischemic brain tissues]

AIM:To detect the expression of TNF-alpha and IL-1beta in human cerebral ischemic tissues. METHODS: 13 cerebral specimens from patients died of cerebral infarction were divided into three groups, <2 d, 3-5 d, and >5d, according to the lasting time of infarctions. The expression of TNF-alpha and IL-1beta in the cerebral ischemic tissues were examined by immnohistochemical staining. The contralateral tissues were employed as controls. RESULTS: The expression of TNF-alpha and IL-1beta in the cerebral ischemic tissues were significant higher than those in the contralateral tissues. The focal distribution of the TNF-alpha(+) and IL-1beta(+) cells was identical with the ischemic area. The expression of IL-1beta and TNF-alpha peaked at the 3rd to 5th and 2nd day after ichemia, respectively. There were no significant difference between the ischemic and contralateral brains at the 5th day after ischemia for the expression of TNF-alpha and IL-1beta. CONCLUSION: Our results showed the expression of TNF-alpha and IL-1beta in human strok of infarction were similar to those in animal experiments. It is suggested that TNF-alpha and IL-1beta are involved in cerebral ischemic injury, which will be helpful for developing clinically a novel therapy aiming at cerebral ischemic injury.     

大鼠脑缺血后脑组织tPA和PAI-1蛋白的表达

为了探讨鼠脑缺血后脑内组织型纤溶酶原激活物 ( t PA)及其主要抑制物 - 1型 ( PAI- 1)蛋白的表达。制备了大鼠全脑和局灶性脑缺血模型 ,用免疫组化法检测 t PA和 PAI- 1蛋白的表达。结果发现 ,在全脑、局灶性脑缺血不同时间和正常鼠脑膜、脉络膜、室管膜、血管内皮等部位均有 t PA、PAI- 1免疫阳性信号 ,但小脑、海马区未见该信号。局灶性脑缺血 6 h,缺血侧中心部位的胶质细胞和梗死灶周边的缺血半影区的神经元也显示 t PA强阳性信号 ,但该细胞不表达 PA I- 1蛋白 ;缺血 18h鼠缺血侧未见t PA、PAI- 1阳性信号。本研究提示 ,脑缺血后鼠脑组织和正常脑组织均有 t PA、PA I- 1蛋白表达 ,但全脑缺血后和正常对照鼠t PA蛋白表达无明显变化 ,而局灶性脑缺血 6 h鼠缺血侧比正常侧 t PA蛋白表达增高 ,脑缺血后鼠脑组织和正常鼠 PA I- 1蛋白表达无显著变化     

缺血后适应对大鼠脑缺血/再灌注损伤的影响

目的：探讨缺血后适应对大鼠脑缺血/再灌注损伤的影响。方法:应用线栓法制作大鼠脑缺血/再灌注损伤模型；21只雄性SD大鼠随机分为缺血/再灌注组、夹闭单侧颈总动脉后处理组和夹闭双侧颈总动脉后处理组，每组7只。再灌注48 h，测定脑梗死体积；拔栓后1 h及处死大鼠前进行神经功能测定；梗死即刻、梗死后10 min、术中1 h、拔栓后即刻、每次夹/松颈总动脉时、干预后30 min等15个时点监测脑血流。结果:夹闭单侧、双侧颈总动脉后处理组大鼠脑组织梗死体积与缺血/再灌注组相比明显减小，有显著差异；3组脑血流各个时点方差分析差异无显著，但是夹闭双侧颈总动脉后处理组干预30 min后脑血流百分比较缺血/再灌注组、夹闭单侧颈总动脉后处理组降低9％。手术后1 h 3组神经功能评分P<0.05，差异显著，夹闭单侧、双侧颈总动脉后处理组神经功能缺损均比缺血/再灌注组减轻。结论:缺血后适应能够明显减小梗塞体积，改善大鼠术后1h神经功能评分，可能与缺血后适应调节早期再灌注时血流动力学状态有关。     

人参皂甙Rd对大鼠脑缺血后丘脑远隔损害的保护作用研究

目的:探讨人参皂甙Rd(GS-Rd)对大鼠脑缺血后对丘脑远隔损害的保护作用。方法:成年SD雄性大鼠,线栓法制备大脑中动脉堵塞脑缺血模型(MCAO),随机分为假手术组,MCAO脑缺血组(1、3、5 d),GS-Rd治疗组(5 mg/kg、10 mg/kg),每组五只。GS-Rd治疗组的大鼠在MCAO手术前30 min,腹腔注射5 mg/kg或10mg/kg GS-Rd,之后每天腹腔注射相应剂量。观察并记录每组大鼠的体重变化;收集大鼠丘脑组织,采用Miliplex试剂盒法检测炎症因子TNF-α、IL-1β、IL-16的含量变化。结果:MCAO脑缺血组大鼠的体重较假手术组减轻(P0.05),而炎症因子TNF-α、IL-1β、IL-16的含量增多(P0.05);与MCAO脑缺血组相比,给予10 mg/kg剂量GS-Rd治疗组的大鼠体重增加(P0.05),而且炎症因子TNF-α、IL-1β、IL-16的含量减少(P0.05);但5mg/kg剂量组较MCAO组体重无显著变化(P0.05)。结论:GS-Rd(10 mg/kg)可能通过减少炎症因子TNF-α、IL-1β、IL-16的含量,减轻大鼠脑缺血后对丘脑等远隔脑区的损害。     

Requirements for tumor necrosis factor-alpha and interleukin-1 in limb ischemia/reperfusion injury and associated lung injury.

Ischemia in rat hind limbs followed by reperfusion results in local as well as remote organ (lung) injury characterized by increased vascular permeability (125I-labeled bovine serum albumin leakage) and hemorrhage (51Cr-labeled rat erythrocytes extravasation) in skeletal muscle and lung, together with an associated increased tissue content of myeloperoxidase, reflecting neutrophil accumulation. Within 60 minutes of reperfusion following ischemia, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 plasma levels increased significantly, reaching maximum levels after 2 hours of reperfusion. Polyclonal antibodies to TNF-alpha and IL-1 provided significant protection against vascular injury in both muscle and lung. These results were confirmed by the use of soluble TNF-alpha receptor and IL-1 receptor antagonist. In rat lungs following ischemia and reperfusion, there was immunohistochemical evidence of E-selectin expression in the lung vasculature; this expression was blocked by treatment of animals with anti-TNF-alpha. These data indicate that both local (hind limb) and remote (lung) organ injury after ischemia/reperfusion requires participation of TNF-alpha and IL-1. The cytokines may, in part, be involved in the up-regulation of endothelial adhesion molecules.     

Early T1- and T2-weighted MRI signatures of transient and permanent middle cerebral artery occlusion in a murine stroke model studied at 9.4T

Early reperfusion following stroke results in reduced tissue injury. Paradoxically, restoration of blood flow under certain conditions may also cause delayed neuronal damage (reperfusion injury). The interrelationship of changes in T1, T2 and diffusion weighted images of tissue water were studied in mouse models of permanent and transient focal cerebral ischemia. A sham surgery or either permanent or transient (30 min) middle cerebral artery occlusion (MCAO) were induced in 14 mice. Magnetic resonance (MR) images of the brain were acquired including: T2 maps, T1 maps and diffusion weighted spin-echo images to produce apparent diffusion coefficient of water apparent diffusion coefficient (ADC) maps. Images were collected on average 90 min after MCAO in both the transient and permanent ischemia groups. Scans were repeated at 24h post-occlusion in mice with transient ischemia. Permanent MCAO resulted in decreases in ADC and no significant change in T2 acutely following MCAO. There were increases in T1 compared to sham controls within the ischemic region in mice following either transient or permanent MCAO (P<0.001). In contrast to permanent MCAO, there were increases in T2 (P<0.001) in the infarct area present in the reperfusion phase within 90 min of transient MCAO. There was considerable infarct growth at 24h (P<0.001). This study demonstrates that following either type of occlusion there are early increases in T1 suggesting an elevated water content in the stroke lesion, while only following transient MCAO are there early increases in T2, indicative of early vasogenic oedema with breakdown of the blood-brain barrier.     

白介素10对脑缺血大鼠神经细胞凋亡的作用研究

目的:探讨白介素10(IL-10)对大鼠脑缺血梗死灶周围神经细胞凋亡的作用。方法:成年雄性Sprague-Darley大鼠36只,随机分为假手术组(Sham组)、局灶性脑缺血组(MCAO组)和脑缺血 IL-10干预组(IL-10组),术后24h断头取脑,TUNEL法(Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling)测定梗死灶周围凋亡神经细胞的数目,免疫组化和RT-PCR法检测促凋亡基因Fas、FasL和caspase-3的表达。结果:脑缺血诱导神经细胞凋亡显著增多(P<0.05),Fas,FasL和caspase-3表达显著上调(P<0.05);IL-10干预可显著减少脑缺血神经细胞凋亡(P<0.05),并抑制FasL和caspase-3的表达(P<0.05),而对Fas的表达无明显作用(P>0.05)。结论:IL-10可抑制大鼠脑缺血梗死灶周围神经细胞凋亡,其机制可能与抑制促凋亡基因FasL和caspase-3的表达有关。     

Zileuton Reduces Inflammatory Reaction and Brain Damage Following Permanent Cerebral Ischemia in Rats

5-Lipoxygenase inhibitor zileuton has been demonstrated to attenuate ischemic brain damage in rats of permanent focal cerebral ischemia in previous work. To further investigate the mechanism underlying zileuton's neuroprotection, adult male Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (MCAO), then received treatment with zileuton or vehicle after the onset of ischemia. Neurological deficit, cerebral infarction, and morphological characteristic were measured 6 and 24 h after MCAO. The enzymatic activity of myeloperoxidase (MPO) was assessed 6 and 24 h after MCAO and the lipid peroxidation levels were evaluated by malondialdehyde assay. Expression of nuclear factor-kappa B (NF-κB) p65 in rat brain was detected by immunohistochemistry and Western blot. Expression of inducible nitric oxide synthase (iNOS) in rat brain was determined by RT-PCR and Western blot. Nitric oxide production in rat brain was also measured 24 h after MCAO. The concentration of TNF-α and IL-1β in serum were detected by ELISA. Zileuton significantly reduced neurological deficit scores, cerebral infarct volume, MPO activity, and the lipid peroxidation levels. It also inhibited the expression of NF-κB and decreased the expression and activity of iNOS in rat brain. In addition, zileuton attenuated the release of TNF-α and IL-1β in serum. Our results suggest that zileuton reduces inflammatory reaction and brain damage in a rat model of permanent focal cerebral ischemia. The neuroprotective effect of zileuton in cerebral ischemia might be associated with the inhibition of inflammatory reaction.     

Suppression of oxidative neuronal damage after transient middle cerebral artery occlusion in mice lacking interleukin-1

Interleukin-1 (IL-1) contributes to ischemic neurodegeneration. However, the mechanisms regulating action of IL-1 are still poorly understood. In order to clear this central issue, mice that were gene deficient in IL-1alpha and beta (IL-1 KO) and wild-type mice were subjected to 1-h transient middle cerebral artery occlusion (tMCAO). Expression levels of IL-1beta and IL-1 receptor I (IL-1RI) were then examined. Generation of peroxynitrite and the expression of mRNAs for nitric oxide synthase (NOS) subtypes were also determined. Immunostaining for IL-1beta was increased from 6 h and peaked at 24 h after tMCAO in the microglia and macrophage. The immunoreactivities of IL-1RI were increased progressively in the microvasculature and neuron-like cells of the ipsilateral hemisphere. Infarct volumes were significantly lower in IL-1 KO mice compared with wild-type mice 48 h after tMCAO (P<0.01). The immunoreactivities of 3-nitro-L-tyrosine were determined in the neurons and microvasculature 24 h after tMCAO and were significantly decreased in the IL-1 KO mice compared to wild-type mice. In addition, expression levels of NOS mRNA in IL-1 KO mice were lower than that measured in wild-type mice. These results indicate that IL-1 is up-regulated and may play a role in neurodegeneration by peroxynitrite production during ischemia.     

大鼠脑缺血再灌注损伤血清IL-8和中性粒细胞趋化指数的变化

目的：探讨了血清IL-8和中性粒细胞趋化指数测定在脑缺血再灌注损伤中的变化。方法：采用改良Zealonga线栓法大鼠大脑中动脉闭塞（ middle cerebral artery occlusion, MCAO）模型,将大脑中动脉（MCA）先行闭塞2h,然后进行灌注不同的时间。应用免疫法测定实验组和对照组血浆中性粒细胞趋化指数,ELISA法测定IL-8。结果：血浆中性粒细胞趋化指数和IL-8于再灌注1h最高,随后缓慢下降,再灌注48h降至正常组水平。结论：中性粒细胞参与了脑组织的正常代谢及生理功能,又参与了脑缺血再灌注损伤的病理生理过程。     

脑的不对称性影响大鼠局灶性脑缺血的结局

为评价左右侧大脑中动脉闭塞(MCAO)对右利大鼠神经行为功能和脑梗死体积的影响,本研究应用四足动物觅食实验筛选右利爪雄性SD大鼠24只,随机分为经左、右侧插线组各12只,8%水合氯醛腹腔注射(300mg/kg)麻醉,线栓法经左、右侧颈外-内动脉插入头端涂有多聚赖氨酸的4-0尼龙线,建立大鼠MCAO缺血2h模型,再灌注72h后评价动物的神经行为功能,测量脑梗死体积。结果表明,所有动物在脑缺血2h神经功能缺损评分最高,再灌注1、24、48和72h经左侧MCAO大鼠显著高于经右侧MCAO大鼠(P<0.05),后者功能明显优于前者,脑梗死体积经左侧插线的大鼠显著大于经右侧插线的大鼠(P<0.05)。研究结果提示,大鼠主侧半球大脑中动脉缺血后,神经功能缺损和脑梗死体积较对侧严重,脑的不对称性影响大鼠局灶性脑缺血的最终结局。     

Neuregulin attenuated cerebral ischemia-Creperfusion injury via inhibiting apoptosis and upregulating aquaporin-4

It has been demonstrated that neuregulin-1beta (NRG-1beta) plays a neuroprotective role in cerebral ischemic injury, however, its defined mechanisms and the perfect treatment window are still elusive. Therefore, we established the animal model of MCAO/R to evaluate cerebral damage. As a result, neurological deficit scores were increased, and a small infarction focus could be seen in ischemic cortex in the control group at ischemic 0.5h/reperfusion 24h. With the duration of ischemia time, deficit scores and infarction sizes obviously elevated in the control group. A large number of positive-apoptotic cells were widespread in the ischemic cortex. Simultaneously, the expression of AQP-4 mRNA and its protein increased. NRG-1beta significantly improved neurological function, decreased the infarction volume, and elevated the expression levels of AQP-4 compared with that in the control group. The therapeutic effect of NRG-1beta was notable, especially at the ischemic 1.0h. These results demonstrate that NRG-1beta might play a neuroprotective effect on cerebral ischemia and reperfusion by inhibiting mitochondrial apoptosis pathway and regulating the activation of AQP-4. The perfect treatment window is at ischemic 1.0h after MCAO.