改良墨汁灌注法与von Willebrandfactor免疫荧光法在大鼠视网膜微血管形态显示中的对比研究

目的:比较改良墨汁灌注法与von Willebrand factor(vWf)免疫荧光法在大鼠视网膜微血管形态显示方面的异同.方法:成年健康SD大鼠随机分成vWf免疫荧光显色组和改良墨汁灌注组.改良墨汁灌注组分两步灌注明胶墨汁40 ml.vWf免疫荧光显色组常规灌注生理盐水40 ml.视网膜行铺片和切片,观察vWf免疫荧光法和改良的墨汁灌注法显示的微血管形态;两组视网膜切片还进行NeuN、Parvalbumin和GFAP的免疫组织化学显色.结果:vWf免疫荧光法能充分显示视网膜铺片周嗣部的微血管,但对铺片中央部和切片的微血管显示不良;改良墨汁灌注法能充分显示大鼠视网膜铺片和切片的微血管,用此方法灌注的视网膜切片的NeuN、Parvalbumin和GFAP免疫组织化学效果均与正常视网膜相似.结论:改良墨汁灌注法在大鼠视网膜微血管形态显示中优于vWf免疫荧光法,且能在同一切片上准确显示血管与神经元/胶质细胞的空间关系.     

加温灌注明胶墨汁制作动物脑血管模型的方法

许多课题的研究都涉及制作动物血管模型，实验中较困难的是既要固定好组织，同时又要在血管内填充染料显示血管，以便在组织学切片染色后，好观察不同脑区的神经核团及血管的分布。我们在对100多只蒙古种沙土鼠进行缺血实验中经过反复摸索，总结出用明胶墨汁温热灌注法，显示动物大脑脑血管的分支分布情况。明胶墨汁灌注动物血管后，取脑组织作冰冻切片，用Nissl染色。镜下观察，神经元着色佳，同时可清楚看到微血管象树枝样纵横交错，相信该方法对脑血管方面的研究技术能提供一定的帮助。     

SD大鼠视网膜中央动脉的应用解剖学研究

目的　为建立稳定可靠的视神经损伤动物模型寻求解剖学依据。 方法　用红色乳胶灌注技术显示正常SD大鼠视网膜中央动脉的来源、分支、分布及其与视神经的关系,并采用体视显微镜摄片测量;明胶墨汁灌注技术显示距眼球后极2.0 mm或6.0 mm处横断视神经后视网膜的血供。 结果　视网膜中央动脉及其分支在视神经鞘内始终与视神经干伴行,视网膜中央动脉起始部到眼球后极的距离为(5.784±0.054)mm;距离眼球后极6.0 mm处鞘内视神经横断组大鼠视网膜单位面积血管数目高于其他部位横断组。 结论　在制备视神经损伤SD大鼠模型时,损伤视神经应在鞘内进行,损伤部位距眼球后极　6.0 mm最佳。     

大鼠视神经内微血管的三维构筑

目的:研究大鼠视神经内血管分布规律.方法:用10%明胶-墨汁混合液经心灌注SD大鼠后取出其视神经,OCT包埋,20μm连续切片,Olympus图像处理系统拍照,用Photoshop 9.0 cs对图像进行处理并用Image-J软件进行分析.用Amira 3.1.1软件将二维切片图像重建成三维可视化图像.结果:三维重建及图像分析显示大鼠视神经内血管围绕神经束排列,球后0～5 mm、5～8 mm、8 mm至视交叉的视神经内血管网形度大于0.5的百分比分别为(55.5±2.7)%、(67.4±4.6)%、(76.8±1.7)%.结论:大鼠视神经内的微血管数量多,走行变化大,从球后到视交叉沿视神经轴由横行逐渐变为纵行.     

糖尿病视网膜病变早期血管内皮生长因子作用机制的研究

目的:通过链脲佐菌素(streptozocin,STZ)糖尿病大鼠视网膜的石蜡切片及血管铺片,研究糖尿病大鼠视网膜病变时血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)作用的分子病理机制。方法:建立糖尿病大鼠模型,随机分为正常对照组(C),糖尿病1月组(M₁)、3月组(M₃)、5月组(M₅)。分别在视网膜石蜡切片及血管铺片上行VEGF原位杂交和免疫组化。结果:①石蜡切片:原位杂交仅M₅表达为67%;免疫组化M₃表达为34%,M₅表达为89%。②视网膜血管铺片:原位杂交仅M₅表达为34%;免疫组化仅M₅表达为56%。结论:①除内皮细胞外,周细胞及Müller细胞也可产生VEGF;②糖尿病视网膜病变早期VEGF可能是来源于旁分泌途径。     

大鼠纹状体血管构筑及增龄变化

目的:研究纹状体血管构筑及其微血管的增龄变化,为探讨纹状体易卒中机制、揭示脑血管病发病机理提供参考资料。方法:运用墨汁明胶灌注、血管铸型、透明、透射电镜等方法观测了三组不同月龄Wistar大鼠纹状体的血管。结果:大鼠纹状体主要由颈内动脉、大脑中动脉、大脑前动脉供血,它们的中央支多以直角发出。尾壳核内部微血管网稠密,与神经纤维、神经细胞之间形成蜂巢状结构。苍白球血管网较稀疏,内囊血管网较苍白球更为稀疏,三者间界限清晰。>24月龄大鼠纹状体毛细血管基膜增厚、内皮细胞固缩、线粒体、内质网等细胞器减少。结论:大鼠纹状体的血供与人的相似,其特殊的形态及增龄变化可能与该区易发生脑血管病有关。     

亲骨荧光素间接标记软骨内成骨的早期血管新生北大核心

背景:虽然当前有多种方法可以观察和检测软骨内成骨早期血管新生,但是均存在一定的不足。目的:探讨四环素和茜素络合酮间接标记软骨内成骨过程中新生血管的可能性。方法:制备新西兰兔双侧桡骨骨缺损模型并植入β-磷酸三钙材料,分别于术后第1天和第15天注射四环素和茜素络合酮,第28天取材。部分标本在墨汁灌注后行硬组织切片荧光/光学显微镜观察,部分标本经脱钙后行免疫组化染色观察,比较软骨内成骨过程中亲骨荧光素标记管腔结构与免疫组化、墨汁灌注标记血管结构的一致性。结果与结论:(1)亲骨荧光素标记的管腔结构经免疫组化染色证实为CD34阳性的血管结构;(2)在荧光显微镜下,亲骨荧光素标记的血管形态与墨汁灌注的血管形态一致;荧光素标记后经墨汁灌注的血管在荧光显微镜下可见荧光管腔中有黑色墨汁走行;(3)此外,亲骨荧光素标记的管腔结构颜色绚丽、三维结构更加生动,可通过不同颜色的荧光显示不同时期的血管新生和演变过程,具有独特的优势,可用于软骨内成骨早期血管发生的形态学检测。     

人皮肤微循环血管形态显示方法的探讨

通过对显示人皮肤微血管各种方法的优缺点的比较，为微循环形态学研究提供资料。进行比较的方法有：墨汁灌注方法、铅丹乳胶灌注方法、A．B．S血管铸型方法和甲基丙烯酸甲酯血管灌注方法。结果：用于显示血管密度，墨汁灌注石蜡切片仍不失为一种较好的方法，而用A．B．S和甲基丙烯酸甲酯这两种充填剂进行血管铸型则各有优缺点。研制既能保持微血管原型又不污染环境的新型铸型剂是一项重要而现实的工诈。     

rAAV2-NTF2对糖尿病大鼠血视网膜屏障的保护作用

目的： 观察2型重组腺相关病毒－核运输因子2（rAAV2-NTF2）对糖尿病大鼠血视网膜屏障功能损伤的拮抗作用。方法: 伊凡思蓝（EB）灌注铺片观察糖尿病大鼠视网膜血管分布和形态。亚克隆构建pSNAV－NTF2载体, 腺相关病毒包装形成rAAV2-NTF2。成年雄性Wistar大鼠,以右眼为实验眼,玻璃体腔注射4 μL rAAV2-NTF2,左眼为对照眼,玻璃体腔注射rAAV2-增强型绿色荧光蛋白（EGFP）4 μL。1个月后取4只大鼠左眼,固定后取视网膜制备全视网膜铺片,荧光显微镜下观察EGFP表达情况。再行STZ腹腔注射诱导糖尿病,同时设单纯糖尿病组（diabetes,D组）和正常组（normal,N组）。糖尿病1个月后以45 mg/kg剂量静脉注射EB,循环2 h后1%多聚甲醛枸橼酸缓冲液灌注并摘除眼球取视网膜,置于甲酰胺中浸泡提取染料,用分光光度计测量甲酰胺中染料的浓度。结果: 正常组大鼠可见在低背景荧光下视网膜血管对EB有很好的屏障作用,糖尿病2周大鼠视网膜血管仅见背景荧光增强,糖尿病1月后血管出现异常节段性扩张,局部血管周围EB渗漏。糖尿病1个月大鼠视网膜内EB浓度是正常组的4.67倍,P<0.01,提示糖尿病大鼠血-视网膜屏障受损。玻璃体腔注射rAAV2-NTF2,视网膜内EB浓度为(8.30±4.16)μg/g视网膜干重,较对侧眼(22.13±8.43)μg/g视网膜干重明显减少,可以部分改善血-视网膜屏障的破坏,降低EB-白蛋白渗漏（P<0.05）。结论: 玻璃体腔注射rAAV2-NTF2可减轻糖尿病大鼠视网膜血-视网膜屏障功能的破坏,为糖尿病视网膜病变的基因治疗提供了实验依据。     

Nestin 和GFAP在缺氧大鼠视网膜神经胶质细胞中的表达及高氧治疗的作用

目的： 探讨巢蛋白（nestin）和胶质纤维酸性蛋白（GFAP）在缺氧大鼠视网膜神经胶质细胞中的表达情况及高氧治疗对其表达的影响。方法: 制作大鼠缺氧模型及缺氧后高氧治疗模型,行眼球矢状位切片及视网膜铺片,行GS/nestin、GS/GFAP、GFAP/nestin免疫荧光双标染色。结果: 正常大鼠视网膜中几乎看不到nestin阳性染色,GFAP阳性染色仅位于星形胶质细胞上。缺氧后,在Müller细胞和星形胶质细胞上出现了nestin的表达,GFAP的表达没有明显变化。高氧治疗后,nestin在Müller细胞上的表达明显减弱,但在星形胶质细胞上仍有较强的表达。GFAP仍然只在星形胶质细胞上表达。结论: Müller细胞和星形胶质细胞针对缺氧损伤和高氧处理发生不同的细胞骨架蛋白重塑,这与它们和视网膜神经节细胞以及视网膜血管在解剖和功能关系上的差异有关。     

Improved method of ink-gelatin perfusion for visualising rat retinal microvessels

To visualize completely rat retinal microvessels, the gelatin-ink perfusion condition was systematically optimized using von Willebrand factor (vWf) immunostaining as control. Whether the vessel showed by the new perfusion condition can be used for double label with neurons or glial cells in the same retina was also tested. Our results showed that infusing rats first with 20 ml of 37 degrees C ink plus 3% gelatin at 140% rat mean arterial pressure (MAP), and subsequently with 20 ml of 37 degrees C ink plus 5% gelatin at 180% rat MAP allowed the ink to completely fill the rat retinal microvessels. Rat retinal microvessels labeled by the perfusion method were more in number than that by vWf immunostaining. Moreover, our data, for the first time, displayed that the improved gelatin-ink perfusion had no effect on and caused no contamination to the following fluorogold labeling or immunostaining of retinal neurons or glial cells in the same tissue. These data suggest that the improved gelatin-ink perfusion technique is a superior method for morphological characterization of rat retinal microvessels, compatible to the double labeling of glial cells and neurons, and it extends the practical scale of the classic method.     

缺血再灌注大鼠视网膜组织型纤溶酶原激活物活性与细胞外基质的相关性

目的：探讨缺血再灌注时大鼠视网膜组织型纤溶酶原激活物（tissue-type plasminogen activator，TPA）活性与视网膜微血管外基质降解的相关性。方法：采用眼压升降法造成视网膜缺血后再灌注。实验组分缺血1～2h再灌注1～2h各组。各组视网膜测试TPA的活性，并用免疫组化法对视网膜微血管外基质作Ⅳ型胶原、层粘连蛋白和纤维粘连蛋白染色。结果：缺血再灌注后，大鼠视网膜TPA的活性随缺血和再灌注时间的延长而显著升高；实验组的Ⅳ型胶原、层粘连蛋白和纤维粘连蛋白阳性染色平均单位面积显著地小于正常对照组，阳性染色呈不连续线状的微血管数显著地多于正常对照组。结论：缺血再灌注可引起视网膜TPA的活性升高，使微血管外基质降解，破坏血-视网膜屏障，导致视网膜结构和功能损伤。     

Angiogenesis at the site of neuroma formation in transected peripheral nerve

Investiture of new microvessels within an injured peripheral nerve trunk may determine the success that the local environment has in promoting axonal sprouting and regeneration. We therefore examined microvessel investment of 24 h–14 d proximal nerve stump preparations in rat sciatic nerves. The stumps, later destined to form neuromas, were created by sciatic nerve transection with resection of distal branches to prevent distal reinnervation. Microvessels were studied in the proximal stump in semithin whole mount sections of nerve and by analysis of India ink perfused microvessel profiles. Quantitative image analysis was made of the luminal profiles of vessels perfused with India ink from unfixed sections of the stumps, contralateral uninjured nerves and sham-exposed but uninjured nerves. Evidence of angiogenesis was observed in stumps 7 d after transection, indicated by a rise in the total numbers of perfused microvessels and in the numbers of 2–6 μm diameter perfused microvessels. There was a shift in the histogram of the percentage of perfused microvessels towards the 2–4 μm range and a reduction in the mean microvessel luminal area in the stumps. By 14 d, new microvessels were larger, indicated by an increase in total luminal area. New microvessels were prominent in the epineurial connective tissue or between layers of perineurial cells of former fascicles. Microvessels probably share a battery of trophic signals with other proliferating cellular elements in the milieu of the injured peripheral nerve trunk.     

慢性高眼压大鼠视网膜胶质细胞TNF-α及其受体的表达

目的： 通过研究大鼠慢性高眼压模型视网膜胶质细胞TNF-α及其受体的表达，探索胶质细胞在青光眼视网膜神经节细胞损伤中的作用机制。方法: 用结扎上巩膜静脉联合术后球结膜下注射5-Fu的方法建立大鼠慢性高眼压模型。在模型建立后1月，做眼球冰冻切片，行免疫组织化学染色，在激光共聚焦显微镜下观察视网膜TNF-α-GFAP、TNF-α-OX42 、TNFR-1-GFAP以及TNFR-1-NeuN的双标染色情况。结果: 结扎上巩膜静脉联合术后结膜下注射5-Fu可诱导较长时间稳定高眼压，4周内6只高眼压眼眼压均大于22 mmHg；正常大鼠视网膜未见明显TNF-α以及TNF-R1阳性染色，1个月高眼压大鼠TNF-α在视网膜的表达高于正常对照组，并且有TNF-α-GFAP、TNF-α-OX42的共表达；在慢性高眼压情况下，TNF-R1在视网膜内层的表达也高于正常对照组，并且在视网膜内层有TNF-R1与 GFAP的共表达，但TNF-R1与神经节细胞层NeuN（神经元特异性核蛋白）没有共表达。结论: 在慢性高眼压情况中，活化的视网膜胶质细胞来源的TNF-α可能在神经节细胞损伤中发挥重要作用。     

Astrocyte invasion and vasculogenesis in the developing ferret retina

We studied the time course of astrocyte invasion and blood vessel formation in the developing ferret retina using glial fibrillary acidic protein (GFAP)-immunohistochemistry for astrocytes and isolectin B4 histochemistry for blood vessels. As in other mammals, strongly GFAP positive astrocytes invade the ferret retina from the optic nerve. At birth, strongly GFAP positive astrocytes have reached about 22% of the distance between optic disc and outer retinal edge whereas weakly GFAP positive processes already extend to the edge of the retina. At postnatal days P30–P37 about 82% of the distance between optic disc and outer retinal edge and in the adult 88% of this distance is covered with strongly labelled astrocytes. Superficial blood vessels form from the optic disc. They reach up to about 24% of the retinal radius at birth and grow radially across the retina during further development. At P30–P37, the whole retina is covered with superficial blood vessels. The deep vascular layer forms later (around P30) through sprouting from superficial vessels. The radial pattern of astrocyte and vessel growth from the optic disc is not affected by the formation of the area centralis and visual streak.     

Perturbation of the blood-retinal barrier after enzyme perfusion. A cytochemical study

The retina is protected from circulating molecules by a blood-retinal barrier. This is comprised of the impermeable apical-lateral junctions of the retinal pigment epithelium and intraretinal blood vessels lined by endothelia that have impermeable junctions and vesicles that do not transport material from the luminal to abluminal front. This study examined the effect of enzyme digestion upon the restrictive properties of the retinal capillary endothelium. Rats were perfused first with enzymes and then by hemoglobin that was visualized by ultrastructural cytochemical methods. After perfusion of buffer alone or buffers containing neuraminidase or heparinase, the cytochemical reaction product was confined to the capillary lumina and to endothelial cell vesicles facing the luminal front. In contrast, after heparitinase or pronase perfusion, reaction product filled the extravascular spaces. Chains of endothelial cell vesicles and patent transendothelial channels were often encountered. Endothelial cell junctions did not appear to be affected by enzyme treatment. These findings indicate that a cell-surface heparan sulfate proteoglycan (or a nonidentified protein removed by proteolysis) is a key molecule required for the maintenance of the blood-retinal barrier.     

Blood-retinal barrier disruption and ultrastructural changes in the hypoxic retina in adult rats: the beneficial effect of melatonin administration

Reactive changes in astrocytes and Müller cells in the retina of adult rats subjected to hypoxia were investigated. Along with this, the integrity of the blood-retinal barrier (BRB) was assessed using fluorescent and electron-dense tracers. In hypoxic rats, mRNA and protein expression of glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQ4) were significantly increased. AQ4 immunoreactive cells were identified as astrocytes and Müller cells by double immunofluorescence labelling. Another alteration in the hypoxic retina was marked reduction in melatonin content compared to controls. In this connection, administration of exogenous melatonin reduced the tissue concentration of vascular endothelial growth factor (VEGF) and nitric oxide (NO); both were elevated in hypoxic rats. A major structural change in the hypoxic retina was swelling of astrocyte and Müller cell processes but this was noticeably attenuated after melatonin administration. Following an intraperitoneal or intravenous injection of rhodamine isothiocyanate (RhIC) or horseradish peroxidase (HRP), leakage of both tracers was observed in the retina in hypoxic rats but not in the controls, indicating that the functional integrity of the BRB is compromised in hypoxia/reoxygenation. It is suggested that enhanced tissue concentration of VEGF and NO production in the hypoxic retina contribute to increased permeability of the retinal blood vessels. The concurrent up-regulation of AQ4, a water-transporting protein, in astrocytes and Müller cells in hypoxia suggests its involvement in oedema formation. Since melatonin effectively reduced the vascular permeability in the retina of hypoxic rats, as evidenced by reduced leakage of RhIC, we suggest that its administration may be of potential benefit in the management of retinal oedema associated with retinal hypoxia.     

Optimal perfusion of an intact ovary as a prerequisite for successful ovarian cryopreservation

BACKGROUND: Cryopreservation and subsequent reimplantation of intact ovaries from cancer patients, offers potentially the best prognosis for restoring fertility after sterilizing cancer treatment. We used bovine ovaries as a model system to explore the perfusion procedure that is required for cryopreservation of intact ovaries. METHODS: The arteria ovarica was cannuled, and ovaries were flushed with Indian ink for 5 min. RESULTS: Successful perfusion of blood vessels was immediately visible macroscopically by a grey to black discoloration of the ovary and was confirmed microscopically, by examining tissue sections. There was no correlation between the time interval from removal of the ovary to the start of the perfusion, and success of perfusion. We determined the percentage of Indian ink-perfused vessels and scored blood vessels in four different size classes. The percentage of perfused vessels increased with an increase in vessel size. In a limited set of preliminary experiments with human ovaries, comparable results were obtained. CONCLUSIONS: Our results show that bovine ovaries are a suitable and adequate model system for optimizing the cryopreservation of human ovaries. As bovine are at least of comparable size to human ovaries, we expect that our results can be extrapolated to the human situation.     

激光损伤视网膜后神经元凋亡及GFAP表达的研究

目的 观察兔视网膜激光损伤后神经元细胞有无凋亡改变及视网膜Ｍｕｌｌｅｒ细胞胶质纤维酸性蛋白（ＧＦＡＰ）的表达变化。方法 应用末脱氧核苷酸转移酶介导的ｄＵＴＰ－Ｘ切口末标记法（ＴＵＮＥＬ法）标记凋亡细胞。应用免疫组化染色显示视网膜Ｍｕｌｌｅｒ细胞ＧＦＡＰ表示。结果伤后６ｈ、１、３、７ｄ视网膜各层可见散在分布的ＴＵＮＥＬ阳性凋亡细胞,尤以外核层多见,伤后３ｄ,视网膜可见Ｍｕｌｌｅｒ细胞ＧＦＡＰ表达;伤