一种媒染血管的新方法—单宁酸—氯化铁法

单宁酸 氯化铁联用媒染组织中的糖蛋白早在1 896年已有文献报道 ,此后许多学者在探讨单宁酸媒染机制及拓宽其使用范围方面做了大量工作[1～ 5] 。实验已证明 ,单宁酸与生物体内糖蛋白、胶原、硫酸粘蛋白 (乙酰肝素、硫酸软骨素、硫酸皮肤素 ) [6 ] 、碳水化合物以及神经组织中的大、小致密核心小泡内的肽类、单胺类递质[3 ,4 ] 等均有较高的亲和性。各级血管壁内亦含有大量糖蛋白、胶原[7] 等物质 ,因此 ,我们设想用单宁酸这种理化特性来显示血管形态也应当没有问题的 ,于是用不同浓度的单宁酸灌流血管 ,组织切片后与不同浓度的氯化铁进行…     

应用单宁酸的负染色效果显示大鼠生殖器官微血管

近百年来,许多学者为研究器官内血管立体构筑倾注了大量心血,创造了多种显示微血管构筑的方法,诸如墨汁灌注法,橡胶、维尼龙血管、淋巴管立体铸型法,微血管铸型扫描电镜法,碱性磷酸酶法（钙铅法）以及单宁酸-氯化铁媒染法（TA-Fe法）,为科学研究及临床应用做出了贡献。继TA-Fe法媒染微血管后,我们又发现单宁酸过度媒染某些组织结构,有类似电镜标本负染色技术效果,清晰地显示了大鼠子宫、卵巢、输卵管等器官的血管构筑特点。     

改良单宁酸-氯化铁法媒染子宫血管的实验研究

目的：光镜下观察大鼠子宫各部位血管构筑。方法：应用单宁酸一氯化铁法(TA—Fe法)灌流固定大鼠，取子宫不同位置做冰冻切片，氯化铁显色，常规脱水、透明、封片，显微摄影。结果：子宫壁出现类似电镜负染色效果，组织结构灰黑色，血管双线条状，分支明显，过管壁切面可见内皮细胞或平滑肌；子宫动脉进入肌层分支并形成一、二级血管网和多支环状动脉，环行动脉或二级血管网发出的微动脉和毛细血管进入粘膜，形成密集的三级毛细血管网。结论：经灌流的子宫标本较长时间保存在媒染固定液内，导致血管外结构也被媒染；子宫壁有三级血管网；每个子宫有多支环状动脉；各段子宫血管构筑不尽相同。     

应用单宁酸-金属盐法媒染脾血管的实验研究

用媒染固定液（２％戊二醛、２％多聚甲醛、２％单宁酸混合液配制）灌流大鼠,取脾做２０－４０μｍ的冰冻切片,蒸馏水复温清洗后入２－３％氯化铁溶液内呈色,１％明胶封片,光镜观察结果：低倍镜示染小动、静脉各级分支以及毛细血管管壁均被染成蓝黑色,脾小结处毛细血...     

改进TA-Fe法显示大鼠肾血管和肾小管的研究

目的 光镜下观察改进单宁酸-氯化铁法（TA—Fe法）媒染肾微血管和肾小管的效果。方法 经TA-Fe法媒染的大鼠肾的两组冰冻切片。一组切片用TA—Fe法，湿片拍照；另一组切片用改进TA—Fe法经脱水、透明、封片。显微摄影。结果 两种TA—Fe法均能显示肾微血管和肾小管。改进TA—Fe法使组织结构更清晰．肾血管球毛细血管分叶更明显。肾小囊上皮、毛细血管内皮细胞、足细胞以及球内系膜细胞核亦被良好显示。切片可较长时间保存。但立体感较改进前略差。结论 改进后的TA—Fe法显示肾微血管和肾血管效果更好，适合做回顾性研究。     

功能确定后的HRP细胞内注射标记的三叉神经中脑核神经元的电镜研究

在猫的Ｐｒｏｂｓｔ束纤维内记录并注射ＨＲＰ，确定出支配咬肌肌梭的三叉神经中脑核神经元，对其进行了连续切片电镜观察．发现约有１０个不明来源的终扣与被标记的中脑核神经元密切接触，其中４个与之形成明确的突触．突触有两种类型：对称性含混合透明小泡（圆形、椭圆形和扁平小泡）者和非对称性含圆形透明小泡者．另外后者也含有为数很少、散在分布的小圆形致密芯颗粒小泡．在其它与标记胞体密切接触的终扣内，也可见到不规则分布的致密芯颗粒小泡．本文对上述所见的可能的功能机制进行了讨论。     

大鼠下丘脑室旁核的突触结构

在PVN神经网中,最多见的是轴—树突触,其次是轴—体突触,偶尔可见树—树突触。根据所含的突触小泡不同,可把轴突末稍分为三类:Ⅰ、含有大量的清亮小泡,混有一些小的致密核心小泡。Ⅱ、含有大量圆的清亮小泡,混有一些大的致密核心小泡。Ⅲ、单纯含有大量圆及椭圆的清亮小泡,大小不等。多种不同性质的轴突末稍共同参与PVN的神经内分泌整合过程。     

多种金属离子与单宁酸反应媒染微血管的对比研究

目的　镜下观察单宁酸与金属盐溶液中Ca2 + 、Au+ 、Ag+ 、Pb2 + 、Cu2 + 、Al3 + 、U6+ 、K+ 联用显示大脑微血管的效果。方法　用单宁酸媒染固定液灌流大鼠 ,取脑切片 ,入氯化钙、氯化金、硝酸银、硝酸铅、硫酸铜、硫酸铝钾、醋酸双氧铀、高锰酸钾和重铬酸钾等溶液中呈色。结果　单宁酸与Ca2 + 、Cu2 + 、Ag+ 、Al3 + 结合显示血管清晰 ,与Au+ 、U6+ 、Pb2 + 、K+ 联用媒染血管效果欠佳。结论　含Ca2 + 、Cu2 + 、Ag+ 、Al3 + 的金属盐类可替代氯化铁媒染微血管 ,氯化金、醋酸双氧铀、硝酸铅、重铬酸钾和高锰酸钾不宜与单宁酸联用来显示血管     

功能确定后的HRP细胞内注射标记的三叉神经中脑核神经元的电…

在猫的Ｐｒｏｂｓｔ束纤维内记录并注射ＨＲＰ，确定出支配咬肌肌梭的三叉神经中脑核神经元，对其进行了连续切片电镜观察。发现约有１０个不明来源终扣与被标记的中脑神经元密切接触，其中４个与之形成明确的突触。突触有两种类型：对称性含混合透明小泡（圆形，椭圆形和扁平小泡）者和非对称性含园形透明小泡者。另外后者也有含有为数很少，散在分布的小园形致密芯颗粒小泡。在其它与标记胞体密切接触的终扣内，也可见到不规则分布     

单宁酸—氯化铁法媒染肾上腺血管的观察

用２％多聚甲醛、２％戊二醛、２％单宁酸配制的媒染固定液灌流大鼠，取肾上腺做冰冻切片放入２—３％氯化铁溶液中呈色，成功地显示了肾上腺皮质、髓质血窦的形貌。高倍镜下可见血窦呈空心管道状，其管壁为细纤维丝状结构编织而成，具有较强立体感。     

大颗粒小泡非突触部位的胞吐——论神经肽释放的另一种形式

本文在以前的工作基础上,进一步用电镜及免疫细胞化学方法,研究了大颗粒小泡非突触部位胞吐作用。实验结果表明,切除大鼠刚髭部皮肤1—24小时之后,术侧延髓后角浅层大颗粒小泡胞吐比对照侧明显增多(P<0.01),术后3—9天复又下降(近似对照动物),术后14—15天又急剧上升(P<0.01)。这些胞吐大部分出现于延髓后角浅层四种轴突终末的非突触部位,少最也发生于树突及轴突中。从术后第6天开始,术侧P物质明显减弱,而甲硫-脑腓肽略有增强。研究结果提示;1)后角浅层胞吐增多,P物质下降及脑腓肽增高,反映了中枢内不同神经元对去传入神经的功能调整作用;2)大颗粒小泡在非突触部位释放神经肽,弥散地作用于远距离的受体,可能起着神经调制物的作用。     

针刺镇痛中大致密核心小泡非突触部位的胞吐及胞吐过程的研究

本文首次采用形态与机能相结合的方法研究了大致密核心小泡(LDV)非突触部位的胞吐和胞吐的动态过程。电镜下观察了在针刺镇痛中三叉神经尾侧脊束核胶状质亚核内出现的各阶段的胞吐影像。据此认为,胞吐是按下列过程连续进行的:1.LDV趋近并伸出一细颈与质膜接触、融合,融合后形成只有一层单位膜的隔,进而隔部分地开放,小的通道形成。2.通道进一步扩大,此时LDV是典型的“Ω”断面像,递质开始排入细胞间隙。3.随通道的进一步扩大,LDV内腔完全开放于细胞间隙,成为间隙的一部分。4.LDV的膜并入终末质膜,仅呈弧形弯曲,其凹侧仍留有少量致密物质,随胞吐物质的弥散,弯曲部分的质膜展平,胞吐的痕迹消失。在针刺镇痛过程中,实验组动物出现的非突触部位胞吐影像比对照组明显增多,两组间的差异非常显著(P<0.01),从而有力地说明与痛觉相关的神经元主要是通过非突触部位胞吐LDV内的递质而参与镇痛过程的。本研究为进一步揭示针刺镇痛机理提供了形态学依据,同时也有力地支持了LDV非突触部位胞吐可能是神经肽释放的主要方式的学说。     

大鼠皮质大颗粒泡的超微结构

用电镜在4只猫听区皮质和3只大白鼠额叶皮质中观察了大颗粒泡的超微结构。在2031个突触面上,共见到大颗粒泡282个。1.大颗粒泡的位置:在104个突触前囊中含有大颗粒泡135个,它们多位于突触前囊的后部(64/135),位于前囊的中部或接近突触前膜者较少。有的大颗粒泡不在突触前囊内,而位于轴突、树突或个别例在树突侧棘中。2.大颗粒泡数目:大脑皮质中的大颗粒泡数目较少。在各种成分中(包括突触前囊、轴突、树突以及其他成分)计见282个大颗粒泡,在一个成分中含1个大颗粒泡的占多数(78.80％)。个别例有含有6个者。多数突触前囊无大颗粒泡。3.大颗粒泡的形态:多为圆形或卵圆形,亚铃形或不整形者也有之。致密核心多数致密,有的并不致密而显得灰淡,有的致密核心呈网状,或在一个包膜内有两个核心。有的膜下环并不明显。4.大颗粒泡的大小不一。本文对大颗粒泡和某些递质的可能关系进行了讨论。     

Noradrenaline release and uptake in isolated small dense cored vesicles from rat seminal ducts

Noradrenaline (NA) release and uptake was investigated in an improved preparation of isolated small dense cored vesicles from rat seminal ducts. The vesicle preparation exhibited an Mg²⁺-ATP-dependent uptake of NA. Half maximal uptake after 20 min was seen at 22 μM. Exchangeability of NA between medium and vesicles was 100%. The kinetics of exchange suggested that mos NA is stored in a single pool. The t1/2 for release of NA was 43 min in the presence of Mg²⁺-ATP, as compared to 15 min in the absence of Mg²⁺-ATP. Addition of reserpine (20 μM) did not significantly alter the NA release. The kinetic properties of this preparation was compared to those of earlier reported noradrenergic vesicle preparations from different tissues.     

Improved isolation of small noradrenergic vesicles from rat seminal ducts following castration. A density gradient centrifugation and morphological study

Noradrenaline storage vesicles from nerve endings have been prepared by differential and density gradient centrifugation. Seminal ducts from castrated rats have been used as a source since fluorescence micrographs and electron microscopy demonstrated an increased concentration of sympathetic nerve endings in this tissue; probably as a result of muscle atrophy. The fractions obtained were analyzed biochemically and partly by electron microscopy.By centrifugation techniques, a density gradient fraction (at the density of 0.4–0.45m sucrose) was obtained in which noradrenaline was enriched about 10-fold from the homogenate. By using organs from castrated rats a further 4-fold increase was obtained in the noradrenaline/protein ratio of the density gradient fraction. This gradient fraction was studied by electron microscopy, after incubation with 5-hydroxydopamine as an exogenous marker, and was found to contain a high proportion of granular vesicle profiles. More than ? of all vesicles with a diameter below about 1000A?had an electron dense core. The vesicle preparation contained low activities of mitochondrial (cytochrome oxidase) and lysosomal (acid phosphatase) marker enzymes, while microsomes and plasma membrane fragments as indicated by NADH-cytochrome c reductase and adenosine triphosphatase had only been partially removed and probably constituted the main contaminants.The distribution of dopamine β-hydroxylase activity suggested that the small, light dense cored vesicles are deficient of this enzyme compared with the large, heavy dense cored vesicles, assumed to occur at the bottom of the gradient. The noradrenaline/adenosine 5'-triphosphate molar ratio was found to be 5.9 in the main noradrenaline fraction, but this fraction was not sufficiently pure to draw any definite conclusions about whether or not noradrenaline is stored together with adenosine 5'-triphosphate in small noradrenergic vesicles.     

与眶下神经相关的初级传入纤维终末的溃变、非突触部位胞吐及突触联系

切断眶下神经的各组大鼠存活2～30天后分别杀死,于其三叉神经尾侧脊束核胶状质亚核内观察了一级传入纤维轴突终末的溃变过程.非突触部位胞吐及突触联系.结果发现:(1)眶下神经的跨节溃变、以突触小泡聚集、融合、空泡形成为主要特征,无微丝增生现象:(2)部分溃变终末内的线粒体明显肿胀变暗.呈球形改变:(3)大致密核心小泡的溃变时间远滞后于突触小泡.两者并不同步进行;(4)轴突终未在溃变过程中,其内的大致密核心小泡仍然进行非突触部位胞吐;(5)溃变纤维终末于胶状质内分别形成轴一树、轴一体、轴一轴三种类型的突触、并参与了突触复合体的形成.     

An electron microscopic study of autonomic nerve cells in the cloacal region of the lamprey,Lampetra japonica

Summary Two types of autonomic nerve cell in the cloacal region of lamprey,Lampetra japonica have been studied by electron microscopy. Large ganglion cells (LGC) were unipolar and individually invested with a satellite cell sheath. The LGC-satellite cell complex measured 24 m × 38 m on average. Granular endoplasmic reticulum and cored vesicles (80–140 nm in diameter) were scattered in the perikaryon. Two kinds of peculiar cytoplasmic filament were seen in LGC: one type was about 20 nm in diameter with periodic dense material on the surface and the other had a diameter of about 8 nm and showed an undulating profile. Nerve endings containing abundant small clear vesicles (30–50 nm in diameter) and a few larger cored vesicles (50–100 nm in diameter) were found in synaptic contact with LGC. Small ganglion cells (SGC) were also unipolar and covered incompletely by a satellite cell sheath. The SGC-satellite cell complex measured 6 and 12 m on average. The SGC was packed with organelles and the perikaryon appeared more electron dense than that of LGC. SGC perikaryonal cytoplasm contained dispersed granular endoplasmic reticulum and numerous large cored vesicles (55–220 nm in diameter). Nerve endings containing numerous large cored vesicles (70–170 nm in diameter) and variable numbers of small clear vesicles (30–50 nm in diameter) were seen on the surface of SGC.     

Application of alcian blue in the electron microscopic study of mouse and human cerebral cortex nerve cells.

Alcian blue is a cationic dye which has been used in the histochemical field for the demonstration of polyanions especially carboxylated and sulphated. The results obtained in neurons when this dye was applied to human and mouse cerebral cortex and studied with the electron microscope are the object of the present report. The CNS of normal adult mice was fixed by vascular perfusion with 2% glutaraldehyde-0.1 M sodium cacodylate-0.1 M sucrose at pH = 6.8 followed by the same fixative with the addition of 0.5% alcian blue. After perfusion, brain cortex was taken out, sectioned into small blocks and immersed in a fresh similar mixture and subsequently in OSO4. Blocks were dehydrated and embedded in araldite. Ultrathin sections were doubly stained with uranyl and lead salts. Human brain cortex taken from patients with cerebral edema was fixed by immersion with 6.5% glutaraldehyde-0.1 M sodium phosphate, pH = 7.4 followed by embedding in warm agar and sectioning in slices of 30 mum thickness which were impregnated by immersion in a mixture of 1% alcian blue-acetate buffer-3% glutaraldehyde at pH = 3.5 for 9 to 15 h at 4 degrees C and subsequently immersed in 1% buffered OSO4-0.1 M sucrose, pH = 7.4 for 2 h at 4 degrees S. Sections were dehydrated and embedded in araldite. Ultrathin sections were doubly stained by uranyl and lead salts. We have denominated the complete procedure in both instances GABOUL technique. The submicroscopic study of both tissues, at nerve cells, revealed the presence of an electron dense homogeneous substance thoroughly dispersed at the hyaloplasmic matrix of perikarya, processes and even synaptic endings. This substance was more evident around free and attached ribosomes, GOLGI apparatus, complex vesicles, dense bodies, microtubules, subsurface cisternae and synaptic vesicles. Canaliculi of endoplasmic reticulum and even the perinuclear cistern also showed a moderate content. It is suggested that this electron dense substance, being alcianophilic, has a polyanionic character and thus may partially correspond to acid polysaccharides since these compounds have already been previously confirmed in such neurons by biochemical and light microscope histochemical techniques.     

三叉神经尾侧亚核大颗粒小泡的非突触部位胞吐及膜再循环的形态观察

在切除大鼠刚髭部皮肤的刺激下,从常规醛-锇酸固定的三叉神经脊束核尾侧亚核的超薄切片中观察到:1.大颗粒小泡轴突终末非突触部位的胞吐影像;2.含致密物质的大有衣小泡,由终末质膜内陷而成,提示大颗粒小泡在终末通过有衣小泡进行膜再循环;3.大颗粒小泡也可由含致密物质的平滑内质网样的管状结构形成。本研究支持大颗粒小泡在非突触部位,通过胞吐释放递质或神经调节物的假说。     

Immunocytochemical analysis of the GABAergic innervation of oxytocin- and vasopressin-secreting neurons in the rat supraoptic nucleus

Antisera specific for gamma-aminobutyric acid (GABA) or its biosynthetic enzyme, glutamate decarboxylase, were used in pre- and postembedding immunocytochemical techniques at the light and electron microscopic levels, to visualize the GABAergic innervation of the hypothalamic supraoptic nucleus. Immunostaining for glutamate decarboxylase or gamma-aminobutyric acid were also combined with oxytocin and vasopressin immunolocalization, thereby permitting evaluation of the contribution of the innervation onto each type of neuron in this nucleus. Light microscopy of semithin plastic sections or vibratome slices stained for glutamate decarboxylase or gamma-aminobutyric acid, with peroxidase-antiperoxidase as immunolabel, revealed an extensive punctate labeling in the supraoptic nucleus and its immediate surroundings. Quantitative analysis of glutamate decarboxylase immunostaining in semithin sections indicated a comparable density of immunopositive punctae at the anterior and posterior levels of the nucleus (14-27 X 10(6) per mm3 tissue). Glutamate decarboxylase- or gamma-aminobutyric acid-immunoreactive cell bodies were never observed within the nucleus although they were detected in the hypothalamus immediately dorsolateral to the nucleus. Electron microscopy of vibratome slices treated with antiglutamate decarboxylase or antigamma-aminobutyric acid and peroxidase-antiperoxidase, or of ultrathin sections stained directly with antigamma-aminobutyric acid and immunoglobulin-coupled colloidal gold, showed that the immuno-reactive punctae represented, in the main, axonal terminals. They invariably contained small, rounded clear vesicles and, at times, one or two larger, dense cored vesicles; they all formed symmetrical synapses onto magnocellular cell bodies and dendrites. Oxytocin and vasopressin neurons were contacted in a similar fashion by glutamate decarboxylase- or gamma-aminobutyric acid-positive boutons in semithin sections of the nucleus stained simultaneously for glutamate decarboxylase and oxytocin and in ultrathin sections stained for glutamate decarboxylase or gamma-aminobutyric acid and oxytocin or vasopressin. Glutamate decarboxylase- or gamma-aminobutyric acid-positive terminals often formed synapses onto two postsynaptic elements in the same plane of section ("double" synapses), a synaptic configuration usually encountered in supraoptic nuclei of lactating animals. In such cases, the postsynaptic somata were oxytocinergic.(ABSTRACT TRUNCATED AT 400 WORDS)