The cholinergic innervation of the rat fascia dentata: identification of target structures on granule cells by combining choline acetyltransferase immunocytochemistry and Golgi impregnation

A monoclonal antibody against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme, was used to study cholinergic synapses on identified (Golgi stained) granule cells in the rat fascia dentata. Choline acetyltransferase immunocytochemistry was applied to 40-microns Vibratome sections cut perpendicular to the longitudinal axis of the hippocampus. Light microscopy revealed fine varicose ChAT-immunoreactive axons in all layers of the fascia dentata, i.e., in the stratum moleculare, the stratum granulosum, and the subgranular polymorph zone. Most fibers were observed in the vicinity of granule cell bodies where they ran mainly parallel to the granular layer. Next, the immunostained Vibratome sections were sandwiched between small pieces of Parafilm and piled to form a block that was covered with agar and Golgi stained. After that, the sections were separated by cutting away the agar and removing the Parafilm. Sections containing well-impregnated granule cells were gold-toned (Fairén et al., '77), embedded in Araldite, and subjected to ultrathin sectioning for electron microscopy. A total of 14 gold-toned granule cells were examined in the electron microscope for synaptic contacts with cholinergic afferents. Choline acetyltransferase-immunoreactive axon terminals were observed that established symmetric synaptic contacts with the cell bodies and dendritic shafts of the gold-toned identified granule cells. Two types of contact were observed on spines arising from gold-toned granule cell dendrites. Immunoreactive terminals established asymmetric synaptic contacts with the head of small spines and symmetric contacts with the stalk of large, complex spines. The boutons forming asymmetric synaptic contacts with the cup-shaped spine head of the complex spines were not found to be immunoreactive. Our results demonstrate that cholinergic fibers to the rat fascia dentata establish characteristic types of synaptic contact with different postsynaptic elements of granule cells, suggesting a complex function of this afferent system.     

The effect of varying impact energy on diffuse axonal injury in the rat brain: a preliminary study

Diffuse axonal injury (DAI) is seen as widespread damage in the white matter of brain characterized by morphological changes to axons throughout the brain and brain stem. The current study attempted to investigate the effect of increasing impact energy on the presence and severity of DAI in corpus callosum (CC). DAI was induced in adult male Sprague-Dawley rats using an injury model adapted from Marmarou et al. in 1994. A 450-g cylindrical brass weight was dropped from three different heights (2.0 m, 1.5 m and 1.0 m) on to a metal helmet affixed to the skull of the rats. In the sham group, rats underwent a surgical procedure with no impact. After a 24-h survival period the animals were transcardially perfused. The brain was removed and the cerebral hemispheres were sectioned with a vibrotome and stained by silver impregnation technique. The CC of all the impacted rats showed DAI in the form of beaded axons, retraction balls and vacuole-like enlargements. The axonal injury was most severe in the 2-m group, while mildest in the 1-m group. In the sham group, axons appeared to be normal. This study demonstrates evidence of graded DAI depending on the impact energy. Such data is useful for mathematical modeling of axonal injury in rat brain using the same impact parameters and potential determination of injury thresholds for neural trauma. Electronic Publication     

Plasticity of innervation of the medulla of axillary lymph nodes in the rat after antigenic stimulation

The purpose of this investigation was to test the hypothesis that activation of the immune system in rats will lead to changes in the density of innervation in lymph nodes. In order to reduce the variability between animals, the rats were reared under sterile conditions and immunostimulation was effected by subcutaneous application of bovine albumin in a region draining to the axillary lymph nodes of both sides. Control animals received an equivalent application of sterile physiological saline. The animals were sacrificed 10 days and 27 days and 4 months after immunostimulation. The nerves in the axillary lymph nodes were quantified by light microscopy in silver impregnated sections and at the ultrastructural level on ultrathin sections. The survival times were chosen so that the first group was in the ascending phase of antibody production, the second group at the peak, and the third group in the declining phase. Both at the light and ultrastructural levels, there were statistically significant differences in the density of innervation of medulla between the groups, with a particularly pronounced increase in the group 4 months after immunostimulation. At the ultrastructural level, there was also an increase in the density of incompletely ensheathed axonal profiles in the parenchyma of the medulla, while the nerves associated with blood vessels were not increased. We conclude that immunostimulation leads to morphological changes in the innervation of the medulla of axillary lymph nodes, that are consistent with the concept of functional activation of the autonomic nervous system through the immune system. © 1994 Wiley-Liss, Inc.     

New and sensitive standard cell culture technique for the detection of cytomegalovirus in clinical specimens

Conventional tube cell culture has been recognised as the most sensitive technique available for human cytomegalovirus (HCMV) detection. Low-speed centrifugation of specimen inocula onto cell culture monolayers has been shown to increase the efficiency of infection with the AD 169 strain of HCMV. Therefore a centrifugal force of 900g for 1 hour at 37 degrees C was used to enhance the detection of HCMV cytopathic effect (CPE) in shell vials that contained circular coverslips with a monolayer of human embryonic lung (HEL) fibroblasts. Of 195 specimens, HCMV CPE was detected in 18 specimens (9.02%) on shell vial culture assay, whereas conventional tube cell culture was positive in only 13 specimens (6.6%). The shell vial culture assay was significantly more sensitive (P less than 0.05). Furthermore the development of the cytopathic effect on shell vial culture assay was significantly earlier (P less than 0.01) and more extensive. Urine samples were sonicated and the results obtained with immunofluorescence using human immune serum demonstrated that sonication increased both the intensity of fluorescence and number of fluorescent foci of HCMV-infected cells and also decreased the non-specific fluorescence of the background.     

The dendritic architecture of the medial terminal nucleus of the accessory optic system in the rat,rabbit, and cat

Summary The neurons of the medial terminal nucleus (MTN) of the accessory optic system (AOS) have been studied in the rat, rabbit and cat in Golgi-Cox and Golgi-Kopsch impregnated brain sections. The present anatomical findings permit a division of the MTN of these species into dorsal and ventral components (MTNd, MTNv), in agreement with other investigations. The MTNd contains predominantly linear-bipolar and linear-multipolar shaped neurons with cell bodies that measure in the range of 25–50 m. These neurons have 2 to 4 primary dendrites which, along with their smaller dendritic branches, are oriented in the plane of the long axis of the MTN (i.e. from ventromedial to dorsolateral). These linear-bipolar and linear-multipolar cells represent 70–80% of the neurons of the MTNd as seen in the Golgi impregnated sections. The remaining 20–30% of the MTNd neurons are nearly all multipolar in shape with somata measuring in the range of 15–25 m. An occasional multipolar neuron is larger, has a soma that measures around 30–60 m and has dendrites which extend outward from the cell body to cover large areas of the MTNd. There was considerable extension of the dendrites of MTNd neurons into the MTNv; however, the dendrites of MTNd neurons were not observed extending into the adjacent substantia nigra (SN) or ventral tegmental area (VTA) of Tsai (1925). Conversely, the dendrites of neurons in the neighboring SN and VTA course along the borders of the MTN but only occasionally extend into the MTN. The neuron population of the MTNv consists almost entirely of small-multipolar shaped cells with somata measuring from 15–25 m and dendritic trees resembling those described for multipolar cells of the MTNd. A small number of neurons of the ventral division are medium-multipolar in shape with cell bodies that measure approximately 30–60 m. Typically, these cells have several dendrites which extend ventrally within the MTNv and one or more dendrites that extend either across the MTNv or dorsally into the MTNd. Only a few linear-bipolar and linear-multipolar neurons were observed in the MTNv. The present findings are discussed in relation to anatomical, physiological, and histochemical studies on the MTN.Abbreviations to Figures CP Cerebral Peduncle - DTN Dorsal Terminal Nucleus - LG Lateral Geniculate Nucleus - LP Lateral Posterior Nucleus - MG Medial Geniculate Nucleus - ML Medial Lemniscus - MTNd Medial Terminal Nucleus, dorsal division - MTNv Medial Terminal Nucleus, ventral division - NTO Nucleus of the Optic Tract - PA Anterior Pretectal Nucleus - pn Nucleus Paranigralis - PP Posterior Pretectal Nucleus - Pul Pulvinar - PO Olivary Pretectal Nucleus - RN Red Nucleus - SGS Stratum Griseum Superficiale, Superior Colliculus - SN Substantia Nigra Supported by USPHS research grant EYO3642 from the National Eye Institute     

离心作用测定细胞与材料间的粘附力及其初步应用

目的：介绍一种简单易行的检测细胞与材料间黏附力的方法。方法：本方法的建立基于离心力在转速一定时与半径成正比的原理。医用石膏制备培养皿固定装置，于培养皿底制备材料薄膜（包括有PLGA，PLGA COLI，PLGA COL I FN），待大鼠骨骼肌卫星细胞悬液与其作用一定时间后，一定转速开动离心机。测定未脱落细胞斑的半径R,取均值。结果：上述三种材料表面未脱落大鼠骨骼肌卫星细胞圆斑半径（R）分别为7.96、15.11、26.10mm，三组间有显著性差异。结论：（1）R的变化可反映细胞与材料间粘附力的改变。（2）利用离心作用检测细胞与材料间的粘附力是可行的，尤其适用于同一种细胞与不同材料间粘附力的比较。（3）COL I和FN的使用可以增加大鼠骨骼肌卫星细胞与PLGA间的粘附。     

人睾丸组织中成熟精子的分离方法初探

探索简单、适用的体外分离睾丸组织中成熟精子的方法,以用于男性无精子症病人行卵母细胞内单精子注射受精-胚胎移植为目的。用含不同浓度透明质酸酶的10 %人血白蛋白人类输卵管液培养人睾丸曲细精管组织匀浆2 4 h,再用Percoll梯度离心法洗涤后计算所获各级精子的总数,并设置前后对照和空白对照进行比较。用不同浓度透明质酸酶处理后的4个实验组分别与2个对照组比较,实验组所获得的各级精子的总数高于对照组,有显著差异(P<0 .0 1)。实验组中透明质酸酶浓度分别为5 0、10 0、15 0 u/ ml的10 %人血白蛋白人类输卵管液处理组获得的各级精子总数组间比较,处理前对照和处理后空白对照之间比较,无显著差异(P>0 .0 5 )。而用浓度为2 0 0 u/ ml的10 %人血白蛋白人类输卵管液处理组获得的各级精子的总数与其它浓度处理组之间比较存在统计学差异(P<0 .0 1) ,其所获得的各级精子总数相似,但活动能力较其它浓度处理组低。透明质酸酶结合Percoll梯度离心可从人睾丸曲细精管组织匀浆中分离出较多更纯的成熟精子,但过高浓度的透明质酸酶可能会影响分离出的精子的活动能力。     

快速大量分离血浆低密度脂蛋白亚组分的方法

目的 :研究快速大量分离低密度脂蛋白亚组分的方法。方法 :通过2步超速离心法分离LDL亚组分 ,并用电泳法及电镜观察鉴定2种亚组分。结果 :仅用10h就将LDL亚组分分开 ,并证实经分离的2种亚组分为纯品。结论 :本方法是1种分离LDL亚组分较好方法     

基于免疫磁珠法的不同筛选方案对胎儿有核红细胞富集效果的比较

目的：将密度梯度离心(density gradient centrifugation,DGC)与免疫磁珠法(magnetic-activated cell sorting,MACS)相结合,应用4种不同的筛选方案,对脐血中的胎儿有核红细胞(fetal nucleated red blood cells,FNRBC)进行分离,评估不同策略富集FNRBC的效果。方法：对10例脐血标本(12 mL/例)按4种不同方案进行筛选：DGC;DGC⁺MACS阴性筛选(CD45^-);DGC⁺MACS阳性筛选(CD71⁺);DGC⁺MACS阴性/阳性筛选(CD45-/CD71⁺)。通过细胞计数板计数,K-B染色后显微镜下人工FNRBC计数,从获得总细胞量、FNRBC量和FNRBC比例经t检验后评估不同方案富集FNRBC的效果。结果：(1)与其他3组相比,DGC方案可获得的总细胞量(0.95×10⁷/mL)最多,FNRBC数量(39.87/mL)最多,F...     

Studies of the dendritic tree of wild-type cerebellar Purkinje cells in lurcher chimeric mice

Naturally occurring mutations are valuable tools for the study of neural development, especially when used in conjunction with the techniques of chimeric mouse production. In this study we examine the response of Purkinje cell dendrites to the altered developmental environment found in the lurcher in equilibrium with wild-type chimera. Lurcher (+/Lc) is an autosomal dominant gene that causes the cell-autonomous degeneration of all Purkinje cells of +/Lc genotype. Thus, in +/Lc in equilibrium with +/+ chimeras, only wild-type Purkinje cells survive to maturity. The number of these survivors can vary from less than 10,000 to greater than 100,000. Previous work has shown that the final ratio of presynaptic granule cells to postsynaptic Purkinje cells is increased in lurcher chimeras. On average, therefore, one might expect that each remaining Purkinje cell would experience an increased supply of afferents, and our hypothesis was that dendritic growth and/or sprouting might occur as a result. This proved incorrect and, indeed, the Purkinje cells in the lurcher chimeras show changes of a predominantly atrophic nature. Unusual morphologies are found, including variable branching density, failure of the distal dendrite to reach the pial surface, loss of isoplanarity, and the frequent appearance of large caliber, primary or secondary dendritic branches ending abruptly in "stub ends." Quantitative analysis of Golgi-Cox impregnated material reveals that in lurcher chimeras the Purkinje cell dendritic arbor is reduced by more than 60% compared to wild-type animals. We present possible explanations for this finding and consider several potential implications.