短期间歇低氧暴露对大鼠海马齿状回神经发生的影响

目的:观察不同低氧浓度环境对大鼠海马齿状回神经发生的影响。方法:取健康雄性Sprague-Dawley(SD)大鼠60只,随机分为11%和15%氧浓度干预组,分别包括常氧对照组(C11,C15),低氧4 h组(4H11,4H15)和低氧8 h组(8H11,8H15)。对所有组大鼠的脑组织切片进行BrdU和NeuN免疫荧光染色,计数海马齿状回内新生细胞数量和新生神经元数量。结果:(1)同一氧浓度组间比较:各组大鼠海马齿状回新生细胞数量(BrdU+)、新生神经元数量(BrdU+/NeuN+)以及新生神经元占新生细胞数量的百分比(Pneuron)均无显著性差异。(2)不同氧浓度、相同低氧干预时间比较:C11和C15,4H11和4H15以及8H11和8H15的BrdU+、BrdU+/NeuN+和Pneuron均无显著性差异。结论:在11%或15%氧浓度环境中,每天进行4 h和8 h的低氧干预,连续7d,对大鼠海马齿状回新生细胞的数量和新生细胞的分化无明显调节作用。     

氯化钴和跑台运动对大鼠海马神经发生及低氧诱导因子-1水平的影响

目的:比较氯化钴(CoCl2)所致机体低氧和跑台运动对大鼠海马齿状回新生细胞数量和低氧诱导因子-1(HIF-1)水平的影响.方法:腹腔注射CoCl2和BrdU于动物,运动方式为跑台运动,大鼠海马齿状回新生细胞和HIF-1采用免疫荧光显色显示.结果:小强度跑台运动组大鼠海马齿状回内BrdU免疫阳性(BrdU+)细胞数量显...     

跑台运动及周龄对大鼠海马神经发生的影响

目的探讨运动及其周龄对大鼠海马神经发生的影响。方法将40只雄性SD大鼠随机分为7周龄安静对照组、7周龄小强度运动组、9周龄安静对照组和9周龄小强度运动组。腹腔注射BrdU于大鼠,持续注射6d。采用免疫组织荧光染色技术,检测各组大鼠海马齿状回内新生细胞和新生神经元数量。结果各组动物海马齿状回BrdU免疫阳性(BrdU+)细胞数目为:7周龄安静对照组50.66±10.54,7周龄小强度运动组74.21±17.00,9周龄安静对照组37.62±9.02,9周龄小强度运动组46.01±10.82。各组动物海马齿状回BrdU++NeuN+细胞数目为:7周龄安静对照组22.54±6.76,7周龄小强度运动组33.38±8.26,9周龄安静对照组15.26±4.42,9周龄小强度运动组17.61±3.86。结论运动促进海马齿状回神经发生,随着周龄的增长而减弱。     

姜黄素对癫痫小鼠海马脑损伤的保护作用

目的研究姜黄素对匹罗卡品诱导的癫痫小鼠海马新生神经元和细胞凋亡的影响。方法姜黄素预处理后,小鼠腹腔注射匹罗卡品建立小鼠癫痫模型,应用新生神经元标记物双皮层蛋白(doublecortin,DCX)免疫组织化学染色及TUNEL染色对造模后72h的模型小鼠海马进行检测。结果 DCX免疫组织化学染色结果表明,与对照组相比,模型组及姜黄素处理模型组小鼠海马齿状回DCX阳性细胞明显减少;与模型组相比,姜黄素处理模型组小鼠海马齿状回DCX阳性细胞明显增多。TUNEL染色结果表明,与对照组相比,模型组及姜黄素处理模型组小鼠海马齿状回TUNEL阳性细胞明显增多;与模型组相比,姜黄素处理模型组小鼠海马齿状回TUNEL阳性细胞明显减少。结论姜黄素可能对匹罗卡品诱导的癫痫小鼠海马神经元有保护作用。     

成年大鼠脑损伤后海马新生神经元功能特点的研究

目的：观察弥漫性脑损伤后海马齿状回新生神经元GAD67和Fos蛋白的表达，探讨海马齿状回新生神经元的功能特点。方法：参考Marmarou方法制作大鼠弥漫性脑损伤模型，采用BrdU标记和免疫荧光组织化学方法并结合激光共聚焦显微镜技术观察海马齿状回中BrdU阳性细胞GAD67的表达和二次致伤后Fos蛋白的表达情况。结果：①成年大鼠弥漫性脑损伤后海马齿状回颗粒细胞层中的BrdU免疫阳性细胞可表达GAD67，而位于亚颗粒增生带的BrdU免疫阳性细胞未见GAD67表达；②在病理刺激下，成年大鼠弥漫性脑损伤后齿状回颗粒细胞层内的BrdU免疫阳性细胞可表达Fos蛋白。结论：弥漫性脑损伤后齿状回颗粒细胞层中的新生神经元不仅可以表达神经活性递质而且能够被病理刺激激活，具有与成熟神经元相似的功能特点。     

VEGF受体抑制剂对Ⅰ型糖尿病肾病大鼠足细胞病的治疗作用

目的:探讨血管内皮生长因子(VEGF)受体抑制剂SU5416对糖尿病肾病足细胞病的治疗作用.方法:SPF级SD雄性大鼠共30只,设为正常对照组(NC)、糖尿病肾病模型组(DN)、SU5416治疗组(SU5416),每组10只.DN大鼠由链脲菌素(STZ)诱导形成.SU5416治疗后第8周,分别测定大鼠体重( body weight,BW)、肾重(kidney weight,KW)、血糖( glucose,Glu)、大鼠24h尿蛋白排泄率(24 h UAER)和血肌酐(Scr).免疫荧光技术观察肾脏足细胞标记蛋白nephrin和podocin表达,Real time-PCR技术检测nephrin、podocin和VEGF mRNA水平,并观察肾脏病理变化.结果:与NC组大鼠相比,DN组大鼠BW明显下降,KW增加显著(P＜0.05),24 h UAER、Glu和Scr均明显升高,肾组织VEGF mRNA水平明显升高,nephrin、podocin蛋白水平及mRNA水平明显下降(P＜0.05),肾小球基底膜增厚、系膜基质增生;SU5416治疗后大鼠肾脏病理改善,KW较DN组明显下降,24hUAER明显降低,nephrin、podocin蛋白水平及mRNA水平上调(P＜0.05),但BW、Glu、Scr和VEGF mRNA水平治疗前后无统计学差异(P＞0.05).结论:SU5416治疗能降低DN大鼠24 h UAER、改善肾脏病理表现和上调肾小球足细胞标记蛋白nephrin和podocin水平.表明VEGF受体抑制剂对DN的治疗作用与其足细胞保护有关,抑制VEGF活性可能成为控制DN足细胞病进展的治疗手段之一.     

营养不良可增加幼鼠齿状回颗粒下层的细胞增殖和神经生发(英文)

为了观察营养不良对幼鼠海马齿状回 (DG)和脑室下层 (SVZ)的细胞增殖和神经发生的影响 ,采用 5 -溴 -2 -脱氧尿苷(Brd U)标记结合免疫组织化学方法对脑切片分别进行 Brd U、Tu J1(β tubulin,β微管蛋白 )及 GFAP(胶质纤维酸性蛋白 )反应或双重反应。结果表明 ,营养不良幼鼠齿状回的细胞增殖和神经生发明显高于营养良好的幼鼠而脑室下层的细胞增殖数量在两者却无明显差异。在齿状回 ,新生的细胞中大约有 5 0 %为新生的神经元 ,10～ 2 0 %为神经胶质细胞。本文结果提示 ,幼鼠海马齿状回的细胞增殖和神经生发可能因营养不良而增加 ,这些新生的细胞可能对日后某些海马依赖性行为产生一定的影响     

异氟醚麻醉抑制海马齿状回神经干细胞的增殖及分化

背景：异氟醚由于起效快、苏醒迅速、无积蓄等优点在儿科手术也逐渐被推广使用,然而其安全性还需要进一步观察。 目的：观察异氟醚麻醉对新生大鼠海马齿状回神经干细胞增殖及神经元分化的影响。 方法：将大鼠随机分为异氟醚组和对照组,分别予以异氟醚吸入麻醉和仅吸入空气。分别在给药前及停止给约后对动物予以5-溴脱氧球苷(BrdU)腹腔注射,第2次注射后24 h处死大鼠,获得脑组织,检测BrdU+和脑内神经源性分化因子表达情况。 结果与结论：停止麻醉处理之后进行血糖以及动脉血气检测,发现异氟醚组大鼠PaCO₂出现轻度上升,pH值出现轻微下降,而PaO₂、BE、SaO₂以及血糖水平则均未出现改变。与对照组相比,异氟醚组经异氟醚麻醉之后,未出现明显的缺氧表现,包括紫绀和呼吸抑制等。大鼠海马齿状回神经干细胞大多分布在门区,异氟醚组的BrdU阳性细胞数少于对照组(P < 0.05)。两组大鼠海马齿状回存在大量新生细胞表达NeuroD,门区NeuroD+/BrdU+阳性细胞数明显高于颗粒细胞下区,且异氟醚组显著高于对照组(P < 0.05)。结果证实,异氟醚麻醉会对新生大鼠海马齿状回神经干细胞增殖及神经元分化产生一定的影响,抑制其增殖,并促进其神经元分化。     

Proliferation and differentiation of neural precursors in hippocampus and dentate gyrus after intracerebral hemorrhage in adult rats

目的:观察成年大鼠脑出血后海马齿状回神经前体细胞的增殖与分化,探讨脑出血后神经前体细胞的变化规律.方法:制作大鼠脑出血模型,5-溴脱氧尿嘧啶核苷(BrdU)腹腔注射标记增殖细胞,用免疫组织化学法检测大鼠海马齿状回BrdU、神经元核抗原(NeuN)、胶质纤维酸性蛋白(GFAP)阳性细胞数的变化.结果:正常组和假手术组大鼠海马齿状回均有少量BrdU阳性细胞,脑出血后大鼠各时间段的BrdU阳性细胞均较正常组和假手术组增加,7d组达到峰值后逐渐下降,28 d组仍高于正常组和假手术组.正常成年大鼠海马齿状回可见少量BrdU/NeuN和BrdU/GFAP双标阳性细胞,脑出血后双标阳性细胞数较正常组增加.结论:脑出血后大鼠海马齿状回神经前体细胞增殖明显,且可以向神经元和神经胶质细胞分化.     

VEGF受体抑制剂对I型糖尿病肾病大鼠足细胞病的治疗作用

目的:探讨血管内皮生长因子(VEGF)受体抑制剂SU5416对糖尿病肾病足细胞病的治疗作用。方法:SPF级SD雄性大鼠共30只,设为正常对照组(NC)、糖尿病肾病模型组(DN)、SU5416治疗组(SU5416),每组10只。DN大鼠由链脲菌素(STZ)诱导形成。SU5416治疗后第8周,分别测定大鼠体重(body we ight,BW)、肾重(k idney we ight,KW)、血糖(glucose,G lu)、大鼠24 h尿蛋白排泄率(24 h UAER)和血肌酐(Scr)。免疫荧光技术观察肾脏足细胞标记蛋白nephrin和podoc in表达,Real tim e-PCR技术检测nephrin、podoc in和VEGF mRNA水平,并观察肾脏病理变化。结果:与NC组大鼠相比,DN组大鼠BW明显下降,KW增加显著(P<0.05),24 h UAER、G lu和Scr均明显升高,肾组织VEGF mRNA水平明显升高,nephrin、podoc in蛋白水平及mRNA水平明显下降(P<0.05),肾小球基底膜增厚、系膜基质增生;SU5416治疗后大鼠肾脏病理改善,KW较DN组明显下降,24 hUAER明显降低,nephrin、podoc in蛋白水平及mRNA水平上调(P<0.05),但BW、G lu、Scr和VEGF mRNA水平治疗前后无统计学差异(P>0.05)。结论:SU5416治疗能降低DN大鼠24 h UAER、改善肾脏病理表现和上调肾小球足细胞标记蛋白nephrin和podoc in水平。表明VEGF受体抑制剂对DN的治疗作用与其足细胞保护有关,抑制VEGF活性可能成为控制DN足细胞病进展的治疗手段之一。     

The role of VEGF/VEGFR2 signaling in peripheral stimulation-induced cerebral neurovascular regeneration after ischemic stroke in mice

Ischemic stroke is a major cause of mortality and morbidity worldwide but effective treatments are limited. Strategies to enhance neurovascular remodeling following stroke provide promising opportunities to improve tissue repair and functional recovery. We have previously demonstrated that whisker activity promotes central angiogenesis in rodent models of whisker–barrel cortex stroke. However, the mechanisms involved in the regulation of neurovascular plasticity by peripheral stimulation are not well-defined. Here, we report that angiogenesis and neurogenesis occur concurrently after cerebral ischemia and whisker stimulation in mice. We show that neuroblasts expressing vascular endothelial growth factor receptor 2 (VEGFR2) migrate along the vessels. Blocking VEGFR2 with the selective inhibitor SU5416 (semaxinib) attenuates ischemia-induced regenerative responses and completely prevents whisker stimulation-induced neurovascular remodeling. These results suggest that VEGFR2-mediated signaling plays an important role in promoting post-ischemia neurovascular remodeling and provides a link between angiogenesis and neurogenesis.     

Oxidative stress and apoptosis interact and cause emphysema due to vascular endothelial growth factor receptor blockade

We have previously demonstrated that a failure of pulmonary endothelial cell survival induced by vascular endothelial growth factor (VEGF) receptor blockade results in lung alveolar septal cell apoptosis and emphysema. Because apoptosis and oxidative stress may be pathobiologically linked, we hypothesized that oxidative stress has a central role in alveolar septal cell apoptosis and emphysema induced by VEGF receptor blockade. When compared with control animals, rats treated with the VEGF receptor blocker SU5416 showed increased alveolar enlargement, alveolar septal cell apoptosis, and expression of markers of oxidative stress, all of which were prevented by the superoxide dismutase mimetic M40419. The preservation of lung structure in SU5416+M40419-treated lungs was associated with increased septal cell proliferation, and enhanced phosphorylation of the prosurvival and antiapoptotic Akt, when compared with SU5416-treated lungs. Consistent with a positive feedback interaction between oxidative stress and apoptosis, we found that apoptosis predominated in areas of oxidative stress, and that apoptosis blockade by a broad spectrum caspase inhibitor markedly reduced the expression of markers of oxidative stress induced by SU5416 treatment. Oxidative stress and apoptosis, which cause lung cellular destruction in emphysema induced by VEGF receptor blockade, may be important mediators common to human and experimental emphysema.     

Microflow of fluorescently labelled red blood cells in tumours expressing single isoforms of VEGF and their response to vascular targeting agents

In this work we studied the functional differences between the microcirculation of murine tumours that express only single isoforms of vascular endothelial growth factor-A (VEGF), namely VEGF120 and VEGF188, and the effect of VEGF receptor tyrosine kinase (VEGF-R TK) inhibition on their functional response to the vascular disrupting agent, combretastatin A-4 phosphate (CA-4-P), using measurement of red blood cell (RBC) velocity by a 'keyhole' tracking algorithm. RBC velocities in VEGF188 tumours were unaffected by chronic treatment with a VEGF-R tyrosine kinase inhibitor, SU5416, whereas RBC velocities in VEGF120 tumours were significantly increased compared to control VEGF120 tumours. This effect was accompanied by a reduced tumour vascularisation. Pre-treatment of VEGF120 tumours with SU5416 made them much more resistant to CA-4-P treatment, with a RBC velocity response that was very similar to that of the more mature vasculature of the VEGF188 tumours. This study shows that vascular normalisation following anti-angiogenic treatment with a VEGF-R tyrosine kinase inhibitor reduced the response of a previously sensitive tumour line to CA-4-P.     

Combined inhibition of vascular endothelial growth factor (VEGF), fibroblast growth factor and platelet-derived growth factor, but not inhibition of VEGF alone, effectively suppresses angiogenesis and vessel maturation in endometriotic lesions

BACKGROUND: Angiogenesis represents the crucial step in the pathogenesis of endometriosis, because endometriotic lesions require neovascularization to establish, proliferate and invade inside the peritoneal cavity. To elucidate the role of angiogenic factors, we investigated in vivo whether blockade of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) affects angiogenesis of ectopic endometrium. METHODS: Mechanically isolated endometrial fragments were transplanted into the dorsal skinfold chamber of hormonally synchronized hamsters. Subsequently, we analysed the effect of the VEGF inhibitor SU5416 and the combined VEGF, FGF and PDGF inhibitor SU6668 on angiogenesis of the ectopic endometrium over a time-period of 14 days using intravital fluorescence microscopy. RESULTS: Selective blockade of VEGF resulted in a slight reduction of microvessel density when compared to control animals. In contrast, combined inhibition of all three growth factors significantly suppressed angiogenesis of endometrial grafts, as indicated by a reduced size of the microvascular network and a decreased microvessel density. This was caused by an inhibition of blood vessel maturation. CONCLUSIONS: Vascularization of endometriotic lesions is not solely driven by VEGF, but depends on the cross-talk between VEGF, FGF and PDGF. Thus, the combined inhibition of these growth factors may represent a novel therapeutic strategy in the treatment of endometriosis.     

Differential effects of cell adhesion,modulus and VEGFR-2 inhibition on capillary network formation in synthetic hydrogel arrays

Efficient biomaterial screening platforms can test a wide range of extracellular environments that modulate vascular growth. Here, we used synthetic hydrogel arrays to probe the combined effects of Cys-Arg-Gly-Asp-Ser (CRGDS) cell adhesion peptide concentration, shear modulus and vascular endothelial growth factor receptor 2 (VEGFR2) inhibition on human umbilical vein endothelial cell (HUVEC) viability, proliferation and tubulogenesis. HUVECs were encapsulated in degradable poly(ethylene glycol) (PEG) hydrogels with defined CRGDS concentration and shear modulus. VEGFR2 activity was modulated using the VEGFR2 inhibitor SU5416. We demonstrate that synergy exists between VEGFR2 activity and CRGDS ligand presentation in the context of maintaining HUVEC viability. However, excessive CRGDS disrupts this synergy. HUVEC proliferation significantly decreased with VEGFR2 inhibition and increased modulus, but did not vary monotonically with CRGDS concentration. Capillary-like structure (CLS) formation was highly modulated by CRGDS concentration and modulus, but was largely unaffected by VEGFR2 inhibition. We conclude that the characteristics of the ECM surrounding encapsulated HUVECs significantly influence cell viability, proliferation and CLS formation. Additionally, the ECM modulates the effects of VEGFR2 signaling, ranging from changing the effectiveness of synergistic interactions between integrins and VEGFR2 to determining whether VEGFR2 upregulates, downregulates or has no effect on proliferation and CLS formation.     

Audiogenic Epilepsy in Mice with Different Genotypes after Neonatal Treatments Enhancing Neurogenesis in Dentate Gyrus

Pups of Wistar and KM rats (with predisposition to audiogenic epilepsy) were daily injected with neuropeptide semax (50 mg/kg) or NO-synthase inhibitor L-NAME (50 mg/kg) on days 7-11 of life. Alterations of audiogenic seizures pattern were revealed in rats of both strains at the age of 1 month, while changes in seizure severity were genotype-dependent. Both agents enhance neurogenesis in the dentate gyrus of the hippocampus and the delayed effect in the form of altered seizure pattern seems to be determined by this factor. Genotype-dependent alterations of seizure severity after administration of semax and L-NAME were differently directed. These effects are suggested to be underlined by physiological and biochemical mechanisms not related to the intensity of postnatal neurogenesis.     

Acute intermittent nicotine treatment induces fibroblast growth factor-2 in the subventricular zone of the adult rat brain and enhances neuronal precursor cell proliferation

Over the past years, evidence has accumulated that stem cells are present in the adult brain, and generate neurons and/or glia from two active germinal zones: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus. This study shows that acute intermittent nicotine treatment significantly enhances neuronal precursor cell proliferation in the SVZ of adult rat brain, but not in the SGZ of the hippocampus, and pre-treatment with mecamylamine, a nonselective nAChR antagonist, blocks the enhanced precursor proliferation by nicotine. This effect is supported by up-regulation of fibroblast growth factor-2 (FGF-2) mRNA in the SVZ and the expression of its receptor FGFR-1 in cells of SVZ showing precursor cells profile. It is also demonstrated that the nicotine effect on neuronal precursor proliferation is mediated by FGF-2 via fibroblast growth factor receptor 1 (FGFR-1) activation by showing that i.c.v. pre-treatment with anti-FGF-2 antibodies or with FGFR-1 inhibitor 3-[(3-(2-carboxyethyl)-4-methylpyrrol-2-yl)methylene]-2-indolinone (SU5402) blocks nicotine-induced precursor cell proliferation. This nicotine enhancement of neuronal precursor cell proliferation was not accompanied by an increase in the number of apoptotic cells. Taken together the present findings revealed the existence in the SVZ of the adult rat brain of a trophic mechanism mediated by FGF-2 and its receptor and regulated by nAchR activation. This possibility of in vivo regulation of neurogenesis in the adult brain by exogenous factors may aid to develop treatments stimulating neurogenesis with potential therapeutic implications.     

The vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR-1) modulates Flk-1 (VEGFR-2) signaling during blood vessel formation

Mice lacking the vascular endothelial growth factor (VEGF) receptor flt-1 (VEGFR-1) die from vascular overgrowth, caused primarily by aberrant endothelial cell division (Kearney JB, Ambler CA, Monaco KA, Johnson N, Rapoport RG, Bautch VL: Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. Blood 2002, 99:2397-2407). Because a second high-affinity VEGF receptor, flk-1, produces a positive endothelial proliferation signal, it was logical to ask whether flt-1 affects developmental blood vessel formation by modulating signaling through flk-1. Differentiated embryonic stem cell cultures lacking flt-1 (flt-1-/-) had increased flk-1 tyrosine phosphorylation, indicating that flk-1 signaling is up-regulated in the mutant background. The selective flk-1 inhibitor SU5416 partially rescued the flt-1-/- mutant phenotype, and this rescue was accompanied by a decrease in the relative amount of flk-1 tyrosine phosphorylation. Thus reduced flk-1 signal transduction can partially compensate for the lack of flt-1. The flt-1-/- mutant phenotype was also partially rescued by Flt-1/Fc, a truncated flt-1 that binds and sequesters the VEGF ligand. Taken together, these data show that down-regulation of flk-1 signaling by two different strategies partially rescues the developmental vascular overgrowth seen in the absence of flt-1, and they support a model whereby flt-1 modulates the flk-1 signal at an early point in the pathway.     

Peptide-directed highly selective targeting of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a disorder of the pulmonary vasculature associated with elevated pulmonary vascular resistance. Despite recent advances in the treatment of PAH, with eight approved clinical therapies and additional therapies undergoing clinical trials, PAH remains a serious life-threatening condition. The lack of pulmonary vascular selectivity and associated systemic adverse effects of these therapies remain the main obstacles to successful treatment. Peptide-mediated drug delivery that specifically targets the vasculature of PAH lungs may offer a solution to the lack of drug selectivity. Herein, we show highly selective targeting of rat PAH lesions by a novel cyclic peptide, CARSKNKDC (CAR). Intravenous administration of CAR peptide resulted in intense accumulation of the peptide in monocrotaline-induced and SU5416/hypoxia-induced hypertensive lungs but not in healthy lungs or other organs of PAH rats. CAR homed to all layers of remodeled pulmonary arteries, ie, endothelium, neointima, medial smooth muscle, and adventitia, in the hypertensive lungs. CAR also homed to capillary vessels and accumulated in the interstitial space of the PAH lungs, manifesting its extravasation activity. These results demonstrated the remarkable ability of CAR to selectively target PAH lung vasculature and effectively penetrate and spread throughout the diseased lung tissue. These results suggest the clinical utility of CAR in the targeted delivery of therapeutic compounds and imaging probes to PAH lungs.