Minimum-invasive model of the rat brain focal ischemia

The problem of brain ischemia damage treatment stimulates the search for new effective methods, which are studied on various experimental models. Most of such models possess significant disadvantages, producing hypoxia in the whole organism, being poorly controlled, involving serious traumas, and still being far from true pathological mechanisms accompanying the ischemia development. A model of focal brain ischemia in rats, induced by middle cerebral artery intravasal occlusion, is free of these disadvantages.     

Anti-apoptotic and neuroprotective effects of edaravone following transient focal ischemia in rats

To investigate the effect of an antioxidant edaravone on the apoptotic process, we examined Bax and Bcl-2 immunohistochemical expression and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reactivity. Rat focal ischemia models were prepared by 2 h transient middle cerebral artery occlusion. Edaravone or physiological saline was intravenously administered after reperfusion. After 24 h of reperfusion, infarction volume assessments, Bax and Bcl-2 immunohistochemistry and TUNEL staining were performed as well as neurological evaluation. Cortical cerebral blood flow was not statistically different between the treatment-groups. Edaravone-treated animals showed significantly improved neurological outcome. Total and cortical infarct volumes in the edaravone group significantly decreased. In addition, edaravone-treatment provided a significant reduction in the number of TUNEL-positive apoptotic cells, a decrease in Bax immunoreactivity and an increase in Bcl-2 expression within the peri-infarct area. Edaravone shows an excellent neuroprotective effect against ischemia/reperfusion brain injury through a Bax/Bcl-2 dependent anti-apoptotic mechanism.     

Activation of astrocytes and ischemic damage following the transient focal ischemia

Astrocytes play vital roles not only in the mechanical support of the central nervous system but also in the metabolism of neurotransmitters and in the transfer of nutritive substances to neuron. After ischemic brain injuries, it has been known that gliosis appears around degenerative regions and repairs these regions. Recently, accumulating evidence indicates that overexpression of S-100 protein, astrocyte-derived protein, is detrimental to neuronal cells in various pathological conditions. To confirm the astrocytic activation in cerebral ischemia, we examined immunohistochemical changes in S-100 protein and glial fibrillary acidic protein (GFAP) in the transient focal ischemia. Cerebral infarction determined by hematoxylin-eosin staining was slight on day 1 and further expanded on day 2 and 3. Thereafter, GFAP immunoreactivity was observed in boundary zone of the infarct area at 72 hours after the transient focal ischemia. On the other hand, S-100 protein immunoreactivities were markedly increased at 9 hours after the transient focal ischemia. After the infarct formation, the increase of S-100 immunoreactivity was observed in outside boundary of infarct area. These results suggest that astrocytic activation, which we would like to be called "pre-mitotic S-100 peak (PSP)", precedes the neurodegeneration following the transient focal ischemia, and should be distinguished from so-called gliosis observed in the post-neurodegeneration and GFAP-dependent astrocytic proliferation.     

应激因子对大鼠实验性脑梗死影响的研究

目的:探讨应激因子对大鼠实验性脑梗死的影响.方法:采用束缚加足底自动电脉冲刺激方法制成大鼠应激模型;采用MCAO大鼠局灶性脑缺血模型;通过检测大鼠束缚应激后血浆中应激因子-皮质醇(Cortisol)、白细胞介素-6(IL-6)、肿瘤坏死因子-a(TNF-a)、血小板活化因子(PAF)等应激标志物变化,探讨应激因子对大鼠实验性脑梗死的影响.结果:①应激24h内各时段血中Cortisol、IL-6、TNF-a和PAF的浓度变化显示,应激24h后均明显后增加(P＜0.01),但短期1h段IL-6、TNF-a、PAF变化不明显.②应激1h后脑梗死范围与非应激脑梗死范围比较无显著差异(P＞0.05),应激24h后脑梗死范围大于非应激脑梗死范围并有显著差异(P＜0.01).结论:短暂应激后应激因子只有Cortisol有一定程度增加,但对脑梗死范围影响不大;过度应激导致多种应激因子明显增加,综合因素加大脑梗死范围.     

右美沙芬在大鼠光化学脑缺血中的神经保护作用

AIM: To study the effects of dextromethorphan (Dex) on photochemically-induced focal cerebral ischemia in rats. METHODS: Anesthetized rats undergone 10-min light irradiation on exposed skull after rose bengal injection were pretreated with saline and Dex at 3 doses (12.5, 25, and 50 mg.kg-1, i.p., 15 min before ischemia). The alteration of volume of lesioned cortical region, regional cerebral blood flow (CBF), bcl-2 and bax expression at penumbra area were studied. RESULTS: Dex dose-dependently decreased the infarcted volume (17%, 26%, and 50% reduction, respectively). Pretreatment with Dex at a dose of 50 mg.kg-1 improved the postischemic hypoperfusion compared with the control at 20 and 30 min after lesion (both 31% increase), and also upregulated the expression of anti-apoptosis gene bcl-2. CONCLUSION: Dex protects against ischemic neuronal damage in this model and its effects on CBF and bcl-2 expression may contribute to its neuroprotective action.     

Expression of hsp 27, hsp 60, hsc 70, and hsp 70 by immortalized human proximal tubule cells (HK-2) following exposure to heat shock, sodium arsenite, or cadmium chloride

The expression of hsp 27, hsp 60, hsc 70, and hsp 70 mRNA and protein was determined in immortalized human proximal tubule cells (HK-2) exposed to heat shock, sodium arsenite, or cadmium chloride (CdCl2) under both acute and extended conditions of exposure. It was demonstrated that the HK-2 cells did not exhibit the classic heat-shock response when subjected to an acute physical (heat) or chemical stress (sodium arsenite or CdCl2). Heat stress, elevated temperature at 42.5 degrees C for 1 h, caused a marked increase only in hsp 70 mRNA and protein, but not hsp 27 or hsp 60 mRNA and protein. Similar results were obtained when the cells were subjected to a classic chemical stress of exposure to 100 microM sodium arsenite for 4 h or CdCl2 for 4 h. These findings were in contrast to those found previously with mortal human proximal tubule (HPT) cells, where acute stress by all three stimuli elicited marked increases in hsp 27, hsp 60, and hsp 70 mRNA and protein. It was shown that the basal levels of expression of hsp 27 and hsp 60 in the HK-2 cells were elevated when compared to those found in unstressed HPT cells and that the basal levels were similar to those found in HPT cells under stress conditions. These results suggest that the failure of the HK-2 cells to increase hsp 27 and hsp 60 levels in response to physical and chemical stress is because they already possess elevated basal levels of these proteins. This would indicate that one or more of the genetic events that resulted in the immortalization of the HK-2 cells also elicited a stress response for hsp 27 and hsp 60, but not for hsp 70, stress response family members. Overall, the results suggest that although there are differences in the regulation of the stress response between the immortal HK-2 and mortal HPT cell lines, as long as these differences are recognized, the HK-2 cell line should be a valuable adjunct to study the stress response of the proximal tubule in general and when exposed to environmental pollutants such as cadmium.     

Expression of heat shock protein 70 and its mRNAs during ischemia-reperfusion in the rat prostate

We investigated the expression of heat shock protein (HSP) 70 and its mRNAs during ischemia-reperfusion in the rat prostate. Eight-week-old rats were divided into six groups: a control group, a 30-min ischemia group, and 30-min ischemia+30-min, 60-min, 1-day, and 1-week reperfusion groups (groups A, B, C, D, E, and F, respectively). In vivo real-time blood flow and HSP 70-1 and 70-2 mRNAs and proteins in the prostate were measured using laser Doppler flow meter, real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) methods, respectively. Clamping of the aorta decreased blood flow to 10% of the basal level. The expressions of HSP 70-1/2 mRNAs increased in groups B, C, and D, and decreased in groups E and F. The expression of HSP 70 proteins was increased after a short interval of increase in their mRNAs. Our data indicated that the expressions of HSP 70 proteins and their mRNAs are dramatically changed during ischemia-reperfusion in the rat prostate.     

疏血通注射液对局灶性脑缺血大鼠的治疗作用

目的:观察疏血通注射液治疗局灶性脑缺血大鼠脑梗死的效果。方法:采用自体血栓注入并闭塞大脑中动脉的方法,制备局灶脑缺血模型。假手术组、大脑中动脉闭塞(MCAO)模型组(模型组)大鼠尾静脉注射生理盐水、疏血通治疗组大鼠尾静脉注射疏血通注射液,均0.25mL·kg-1,qd,连续5d。观察3组大鼠的脑梗死范围、脑含水量、神经功能评分、血管闭塞和再通情况、病理组织学改变等指标的变化。结果:模型组大鼠脑含水量(84±s3)%,比假手术组(74±3)%明显增多;疏血通注射液可明显改善神经功能缺失评分,缩小脑梗死范围,降低脑组织含水量,此外疏血通注射液还可促进血栓溶解,使血管再通并减轻脑组织病理变化。结论:疏血通注射液对局灶性脑缺血大鼠急性脑梗死有明显的治疗作用。     

Minocycline inhibits 5-lipoxygenase activation and brain inflammation after focal cerebral ischemia in rats

Aim： To determine whether the anti-inflammatory effect of minocycline on postischemic brain injury is mediated by the inhibition of 5-1ipoxygenase （5-LOX） expression and enzymatic activation in rats. Methods： Focal cerebral ischemia was induced for 30 min with middle cerebral artery occlusion, followed by reperfusion. The ischemic injuries, endogenous IgG exudation, the accumulation of neutrophils and macrophage/microglia, and 5-LOX mRNA expression were determined 72 h after reperfusion. 5-LOX metabolites （leukotriene B4 and cysteinyl leukotrienes） were measured 3 h after reperfusion. Results： Minocycline （22.5 and 45 mg/kg, ip, for 3 d） attenuated ischemic injuries, IgG exudation, and the accumulation of neutrophils and macrophage/microglia 72 h after reperfusion. It also inhibited 5-LOX expression 72 h after reperfusion and the production of leukotrienes 3 h after reperfusion. Conclusion： Minocycline inhibited postischemic brain inflammation, which might be partly mediated by the inhibition of 5-LOX expression and enzymatic activation.     

复方葛根注射液对大鼠局部脑缺血的保护作用及部分机制研究

目的观察复方葛根注射液对大鼠局部脑缺血的保护作用及其作用机制.方法采用大鼠结扎右侧大脑中动脉(Right MiddleCerebral Artery Occlusion,RMCAO)方法造成局灶性脑缺血模型,研究复方葛根注射液在脑缺血中的保护作用.测定脑组织匀浆中的一氧化氮(NO)及一氧化氮合酶(NOS)含量,探讨药物作用机制.结果复方葛根注射液能够明显减轻脑梗死重量,保护神经细胞,降低脑匀浆中NO及NOS含量.结论复方葛根注射液对脑缺血有明显的保护作用,其机制与其降低脑局部的NO及NOS有关.     

Endothelin-1诱导的大鼠局灶性脑缺血再灌注模型

目的　研究Endothelin 1(ET 1)诱导的大鼠局灶性脑缺血再灌注模型。方法　将ET 1注入大脑中动脉 (MCA)附近 ,ET 1强烈的缩血管作用可使脑血流 (CBF)降低造成缺血性脑损伤 ,采用氢清除法监测缺血过程中大鼠纹状体脑血流的变化 ,于缺血后 6h进行神经功能评分 ,并采用TTC染色法观察脑梗死面积的变化。结果　ET 1可使纹状体血流呈现浓度依赖性的降低 ,10min时CBF降至最低 ,分别为 :ET 130 0 pmol组 (2 7 1± 2 9) %、36 0pmol组 (12 7±2 1) %、4 0 0pmol组 (11 9± 1 8) %、5 0 0 pmol组 (9 5±1 6 ) % ;神经功能评分显示ET 1可造成大鼠不同程度的神经功能损伤 ;MCAO后 2 4h ,脑梗死面积百分率随ET 1浓度的增加而呈上升趋势 ,并显示出良好的浓度依赖关系 :30 0pmol组 (3 9± 0 3) %、36 0 pmol组 (7 4± 0 5 ) %、4 0 0 pmol组 (11 3± 1 3) %、5 0 0 pmol组 (16 2± 1 8) % ;r =0 992 6(P <0 0 5 )。结论　该模型操作简单 ,造成的脑梗死范围稳定、重现性好 ,是一种更接近人类卒中具体情况的较理想的局灶性脑缺血再灌注模型     

Ginsenoside Rd promotes neurogenesis in rat brain after transient focal cerebral ischemia via activation of PI3K/Akt pathway

Aim:

To investigate the effects of ginsenoside Rd (Rd) on neurogenesis in rat brain after ischemia/reperfusion injury (IRI).

Methods:

Male SD rats were subjected to transient middle cerebral artery occlusion (MCAO) followed by reperfusion. The rats were injected with Rd (1, 2.5, and 5 mg·kg⁻¹·d⁻¹, ip) from d 1 to d 3 after MCAO, and with BrdU (50 mg·kg⁻¹·d⁻¹, ip) from d 3 to d 6, then sacrificed on 7 d. The infarct size and neurological scores were assessed. Neurogenesis in the brains was detected by BrdU, DCX, Nestin, and GFAP immunohistochemistry staining. PC12 cells subjected to OGD/reperfusion were used as an in vitro model of brain ischemia. VEGF and BDNF levels were assessed with ELISA, and Akt and ERK phosphorylation was measured using Western blotting.

Results:

Rd administration dose-dependently decreased the infarct size and neurological scores in the rats with IRI. The high dose of Rd 5 (mg·kg⁻¹·d⁻¹) significantly increased Akt phosphorylation in ipsilateral hemisphere, and markedly increased the number of BrdU/DCX and Nestin/GFAP double-positive cells in ischemic area, which was partially blocked by co-administration of the PI3 kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Treatment with Rd (25, 50, and 100 μmol/L) during reperfusion significantly increased the expression of VEGF and BDNF in PC12 cells with IRI. Furthermore, treatment with Rd dose-dependently increased the phosphorylation of Akt and ERK, and significantly decreased PC12 cell apoptosis, which were blocked by co-application of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002.

Conclusion:

Rd not only attenuates ischemia/reperfusion injury in rat brain, but also promotes neurogenesis via increasing VEGF and BDNF expression and activating the PI3K/Akt and ERK1/2 pathways.     

大鼠局灶性脑缺血／再灌注后脑组织病理变化的研究

目的:本文旨在观察大鼠大脑中动脉栓塞/再灌注后动物的脑组织含水量、脑梗塞体积和脑组织的病理学变化。方法:采用插线法制作大鼠大脑中动脉栓塞/再灌注模型,并观察脑缺血/再灌注不同时点大鼠的神经病学评分(Kuluz法)、脑组织含水量(Hallenbeck法)、脑梗塞体积(Nedergaard法)、组织病理学变化(HE染色)。结果:大脑中动脉栓塞3h、栓塞3h/再灌注24、48h时,损伤加重。结论:再灌注后脑组织损伤加重。     

Effects of cerestat and NBQX on functional and morphological outcomes in rat focal cerebral ischemia

This study investigated the ability of NBQX, an AMPA receptor antagonist, and cerestat, a NMDA receptor antagonist, to counteract neurological deficits and morphological damage induced by permanent occlusion of the left middle cerebral artery (MCAO model) in the rat. NBQX (3, 10, and 30 mg/kg, ip) injected at 10, 60, and 120 min postocclusion did not reduce the volume of infarct in the MCAO model of cerebral ischemia and had marginal effects on sensory dysfunctions (vibrissae stimulation and body proprioception) and no effects on motor dysfunctions (forelimb flexion and footfault test). Conversely, cerestat (0.3, 1, and 3 mg/kg, sc) injected at 10 and 120 min postocclusion significantly reduced the ischemic volume at the dose of 1 mg/kg, and, at the same dose, significantly attenuated behavioural deficits in the body proprioception and in the forelimb flexion tests.     

Mechanisms of lateralized hyperactivity following focal brain injury in the rat

Recent work in this lab which explores the differential behavioral and neurochemical changes following left versus right cerebral damage is reviewed and neural mechanisms which may account for this asymmetry are proposed. Damage to the right frontoparietal cortex in rats, caused by either ischemia or suction, produces hyperactivity for as long as a month after surgery. These lesions also produce decreases in norepinephrine (NE) levels in both ipsilateral and contralateral cortex and in the locus coeruleus. Similar lesions in the left cortex, however, do not produce these behavioral or biochemical changes. Similar lateralized responses have also been produced by intracortical injections of NE neurotoxins, by cortical island lesions, by destroying cortical efferents, and by producing lesions in the nucleus accumbens, which receives a cortical input. These lateralized responses suggest that the neural mechanisms that mediate this phenomenon include both cortical and subcortical components. It is proposed that the neuroanatomical asymmetry is in either accumbal efferents or their postsynaptic connections.     

Anti-stroke profile of thiazolidin-4-one derivatives in focal cerebral ischemia model in rat

Recently, some PPARγ agonists like pioglitazone, rosiglitazone, and other newer thiazolidine-2, 4-dione (TZD) derivatives have been shown to be neuroprotective in experimental model of cerebral ischemia/reperfusion (I/R) injury. Replacement of active pharmacophore viz: thiazolidine-2,4-dione of these PPARγ agonists with biologically privileged scaffold thiazolidin-4-one derivatives have been synthesized and bioevaluated in focal cerebral ischemia model in rats with an aim to ameliorate cerebral ischemic damage. Of 20 synthesized molecules, three of the substituted compounds (2, 6 and 18) have shown significant (p < 0.001) neuroprotection even much better than rosiglitazone at same dose, when administered 1 h prior to 2/24hrI/R cerebral injury in rats, whereas compounds 10, 15, and 17 also showed significant but moderate effect on most of the parameters used in the study. Moreover, compound 2 and 6 also showed curative potential after 6 h post I/R treatment. The compound 2 has also shown significant effect on glutamate uptake by perhaps enhancing the GLT-1 activity. Thus, the present study indicates that some of the synthesized thiazolidin-4-one substituted PPARγ agonists exhibit better neuroprotection and have potential to ameliorate the ischemic damage. Therefore, this novel class of compounds could be further suitably modified to obtain potent anti-ischemic agents, warranting clinical exploitation.     

A potent antioxidant, lycopene, affords neuroprotection against microglia activation and focal cerebral ischemia in rats

We investigated the effects of a potent antioxidant, lycopene, on the free radical-scavenging activity as evaluated by the DPPH test and lipid peroxidation in rat brain homogenates as well as nitric oxide (NO) formation in cultured microglia stimulated by lipopolysaccharide. In addition, we also investigated the therapeutic effect of lycopene in attenuating ischemia/reperfusion brain injury induced by middle cerebral artery (MCA) occlusion in rats. Lycopene (1, 2 and 5 microM) exerted increased DPPH decolorization in the DPPH test, and increased inhibition of iron-catalyzed lipid peroxidation (TBARS formation) in rat brain homogenates in concentration-dependent manners. Furthermore, lycopene (5 and 10 microM) significantly inhibited nitrite production by about 31% and 61% in microglia stimulated by LPS, respectively. Rats which received lycopene at a dosage of 4 mg/kg, but not at 2 mg/kg, showed significant infarct size reductions compared with those which received the solvent control (20% Tween 80). In conclusion, we demonstrate a protective effect of lycopene on ischemic brain injury in vivo. Lycopene, through its antioxidative property, mediates at least a portion of free radical-scavenging activity and inhibits microglia activation, resulting in a reduction in infarct volume in ischemia/reperfusion brain injury.     

神经节苷脂对大鼠脑缺血再灌注后Caspase-3表达的影响

目的研究单唾液四己糖神经节苷脂（Monosialoganglioside,GM1）对大鼠局灶性脑缺血再灌注损伤后神经功能缺失评分及Caspase-3表达的影响,探讨其神经保护作用机制。方法42只Wistar雄性大鼠随机分为三组,即神经节苷脂GM1治疗组,脑缺血再灌注损伤模型组,正常对照组。采用改良线栓法建立右侧大脑中动脉缺血再灌注模型。治疗组缺血即刻腹腔注射30mg/kg神经节苷脂,模型组注射等剂量生理盐水,运用Zea-Longa法进行神经病学评分,用光镜观察脑组织形态学改变,用免疫组化法检测Caspase-3。结果与模型组相比,GM1治疗组脑组织变性坏死程度轻,神经功能缺损评分显著减少,Caspase-3阳性细胞减少（P〈0.01）;模型组Caspase-3表达明显多于对照组（P〈0.01）,而且缺血再灌注24h较再灌注1h明显增多（P〈0.01）。结论GM1可以通过抑制Caspase-3的表达来抑制细胞凋亡,从而发挥脑缺血再灌注损伤的保护作用。     

Copper and zinc uptake and hsp70 expression in HepG2 cells.

The aim of this work is to study the accumulation in HepG2 cells of two essential metals with toxic potency and to analyse the induction of the heat shock protein 70 kDa (hsp70) consequent to metal exposure. Cu and Zn were the metals considered and were analysed both as single compounds and in combination in order to evidence synergic effects of the mixture. The use of HepG2 cells provided an in vitro system that retains morphological and metabolic properties and the expression of specific genes typical of liver parenchymal cells. Moreover, the hepatic cells represent a suitable model for their susceptibility to metal toxicity since liver, gastrointestinal tract and renal tubular cells are involved in the uptake, transport, detoxification and secretion of these compounds. The uptake of Cu and Zn followed a time-dependent accumulation when they were used separate. The combination of the two metals produced a higher accumulation of Zn. The stress protein hsp70 was expressed before the metals accumulated within the cells, as shown by the measures obtained with the ICP-AES technique. Moreover, the accumulation of hsp70 by a sublethal shock provided a protective mechanism against metal cytotoxicity.