Non-neuronal cells in the spinal cord of nude and heterozygous mice. I. Ventral horn neuroglia

Summary A quantitative light microscopic analysis of the ventral grey matter in the lumbar spinal cord of homozygous nude (nu/nu) and heterozygous (nu/+) mice was performed to determine the possible contribution of lymphocytes to normal C.N.S. tissue. If lymphocytes were present in the neuropil, they could be mistaken for neuroglial cells. Athymic nude mice offer a good model, since they lack T-lymphocytes and symptoms of neurological involvement. Mean cell counts from 1 m sections were tested by analysis of variance. There were no strain differences for the area and number of neurons. The total neuroglial cell count was also similar, but the number of oligodendrocytes decreased 28%, astrocytes increased 51% and microglia were unchanged in the nude compared with the heterozygous mouse. There were no qualitative differences at the ultrastructural level among the neuroglia of either strain. Either the genetic defect retards and alters neuroglial cell development, or some of the small, round dark nuclei belong to lymphocytes, which have earlier migrated into the C.N.S. parenchyma. Lymphocytes could then participate in a cell-mediated immune response with brain macrophages, which are thought to be primarily derived from mononuclear leukocytes.     

Transplantation of Schwann cells to subarachnoid space induces repair in contused rat spinal cord

Schwann cell transplantation is well known to induce repair in the injured spinal cord which disables millions of injured patients throughout the world. An ideal route of delivering the grafted Schwann cells to the spinal cord should neither cause more injury nor reinitiate inflammatory events and also provide a favorable milieu to the grafted cells. In this study, we have utilized subarachnoid route to transplant Schwann cells and evaluated their effects in a contusive model of spinal cord injury. Adult rats weighing 100-140 g were experimentally injured by crushing the spinal cord with a titanium clip and then divided into four groups (Tracing, Control, Medium-treated and Schwann cell-treated). Cultured Schwann cells (5x10(4) cells in 5 microl) or medium were injected to the animals of corresponding groups via subarachnoid space at the injured site 7 days after injury. In tracing group, Schwann cells (labeled with Hoechst) demonstrated their presence within spinal cord 7 days after transplantation. Evaluation of locomotor performance of animals for 60 days after injury showed that animals treated with Schwann cells had significant improvement (P<0.01). Similarly, the axon density at the site of injury was significantly higher. The results indicate the efficacy of subarachnoid route for the transplantation of Schwann cells in inducing repair of the contused spinal cord. We conclude that this route can be useful for the transplantation of Schwann cells and offers a hope for the patients suffering from spinal cord injury.     

Serum immunoglobulins in nude mice and their heterozygous littermates during ageing.

Serum immunoglobulin (Ig) levels were investigated in 6, 40 and 110 week old congenitally athymic (nude) mice and their heterozygous littermates. Concentrations of IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA were determined by rocket electrophoresis. At 6 weeks of age, IgM was the most prominent serum Ig in both nude and heterozygous mice. Except for IgM and IgG3, some nude mice displayed unquantifiable levels of some of the other Ig classes or subclasses. At this age, the average levels of the various Ig classes and/or subclasses did not differ significantly between the two groups of mice. At the ages of 40 and 110 weeks, most nude mice showed serum Ig spectra in which all classes and subclasses were present. Young (6 week) and middle-aged (40 week) nude mice generally showed a wider variation in Ig levels than did their heterozygous littermates. The most striking differences between aged nude mice and aged heterozygous mice were: (a) the generally decreased levels of IgG2a, IgG2b and IgA; (b) the frequent occurrence of increased serum levels of IgG1; and (c) the increased incidence of homogeneous Ig components (''paraproteins'') in the sera of nude mice.     

Chronic catheterization of the spinal subarachnoid space

To administer drugs into the spinal subarachnoid space of unanesthetized and intact rats and rabbits, a procedure is described whereby a polyethylene catheter (PE-10) may be inserted through a puncture of the atlanto-occipital membrane and secured to the skull. Calibration experiments carried out with bromophenol blue dye, 3H-naloxone and 14C-urea revealed first, that there was little rostro-caudal diffusion of the injectate along the spinal axis and secondly, that even for compounds such as naloxone which can rapidly permeate neural tissues, the levels which do appear in the brain are small following the spinal subarachnoid administration of the drug. Control injections, administered either acutely or repeatedly over a prolonged period of time, had no detectable effect on the animal's behavior. These observations, as well as the lack of pathology in the spinal cords of rats having such catheters for periods of up to 4 months suggests that the implant is well tolerated.     

Clearance of some quaternary amines from the spinal subarachnoid space.

The spinal subarachnoid space was perfused with artificial cerebrospinal fluid (CSF) from the low lumbar level to the middle thoracic level or to the cisterna magna in anesthetized rabbits. 3-H-choline, 3-H-methyl-atropine or 3-H-decamethonium with carrier in different concentrations was added to the perfusate together with 14-C-inulin, the latter serving as a marker of the dilution of the perfusate by original CSF. Choline was eliminated from the perfusate partly by a saturable mechanism probably by an uptake into the spinal cord. About 15 per cent of the radioactivity of the choline infused was recovered from the spinal cord mainly as phosphorylcholine, betaine, and phospholipids. Amphetamine decreased the elimination of choline from ventriculocisternal perfusates and partly inhibited the uptake of choline in rabbit choroid plexus in vitro. In contrast, amphetamine did not influence the saturable elimination of choline in the lumbothoracic perfusion. Neither methylatropine nor decamethonium was eliminated from the perfusate by a saturable mechanism in the lumbothoracic perfusions. However, in perfusions including the cisterna magna methylatropine was partly eliminated by such a mechanism. The concentration of radioactivity in fourth ventricular choroid plexa suggested this structure to be responsible for the saturable part of the elimination. In conclusion, there is no active removal of quaternary amines in general from spinal CSF like the choroid plexus mediated clearance from ventricular CSF.     

蛛网膜下腔置管灌注rhNCAM1促损伤脊髓再生修复的实验研究

目的:探讨神经细胞粘附分子1(neural cell adhesion molecule 1,NCAM1)灌注治疗小鼠脊髓撞击伤后对脊髓修复及神经功能恢复的影响。方法:采用改良NYU(纽约大学)Weight-Drop Impactor打击装置,以25 gcf(l0 g重量从25 mm高度自由落下)制备脊髓损伤模型(打击面积为2 mm×2 mm)。将SA小鼠30只随机分为3组:实验组(rhNCAM1组,n=10)于损伤即刻经蛛网膜下腔注入rhNCAM1蛋白2μl(约100μg),隔日1次,共5次;对照组(生理盐水组,n=10)于损伤即刻经蛛网膜下腔注入生理盐水2μl,隔日1次,共5次;假手术组仅做椎板切除术,术后缝合切口。观察各组小鼠1~8周行为学改变,行BBB评分。第8周灌注固定、取材,采用神经纤维200(NF200)免疫荧光检测,进行图像分析及统计处理。结果:(1)行为学评估:实验组BBB评分在各时间点均显著高于对照组(P<0.01);(2)NF200免疫荧光染色:实验组阳性着色明显多于、并强于对照组;实验组无组织瘢痕结构面积明显小于对照组。结论:蛛网膜下腔应用rhNCAM1后可能改善SCI后损伤区及两端的神经细胞之间的功能,促进轴突再生,促进双下肢运动功能的恢复。     

Enhanced phagocytic activity of blood leukocytes in athymic nude mice

After 30-min incubation, blood leukocytes of adult athymic BALB/c nude mice (nu/nu) have a distinctly higher methacrylate or yeast particle uptake than leukocytes of euthymic nu/+ or +/+ mice. This permanent enhancement is not due to humoral factors, since the percentage of phagocytosing nu/nu leukocytes increases further in nu/+ littermate's plasma. Also, chronic infection or intraperitoneal immunization causes an additional transitory increase of the percentage of phagocytosing leukocytes and numbers of particles ingested; the phagocytic performance of leukocytes of euthymic mice is raised under these conditions by a greater factor. In fetuses enhanced phagocytosis of nu/nu mice is found only in monocytes. The bulk of ingestion of methacrylate particles is mediated by Fc receptors and significantly more receptors for IgG2B, IgG1, and IgG2A are demonstrable on nu/nu neutrophils; ingestion of particles via these receptors is again higher in nu/nu neutrophils, whereas nu/nu monocytes display a higher uptake via Fc(IgG1) receptors.     

Cholecystokinin releases [3H]GABA from the perfused subarachnoid space of the anaesthetized rat spinal cord

The neuropeptide cholecystokinin(26-33) (CCK) is widely distributed in the mammalian central nervous system, including the spinal cord. We have studied the possible interaction of CCK with GABA release mechanisms. Low doses of CCK-8 (1 nM) have been found to evoke calcium-dependent [3H]GABA release from an in vivo perfused spinal cord preparation in the anaesthetized rat. Tachyphylaxis was seen to the [3H]GABA releasing action of CCK-8. The injection of proglumide (150 mg/kg i.p.) totally blocked the [3H]GABA release produced by CCK-8 or by a medium containing 50 mM potassium. Substance P (10 microM) did not produce release of [3H]GABA, although in the same animals 50 mM potassium containing solutions could be shown to evoke release of [3H]GABA.     

The ultrastructure of Auerbach's plexus in the guinea-pig. II. Non-neuronal elements

Summary The ultrastructural features of non-neuronal cells associated with Auerbach's plexus in the stomach, ileum and colon of the guinea-pig have been examined. Apart from Schwann, mast and interstitial or fibroblast-like cells, two other cell types are described that do not appear to have been reported previously. Of these two cell types, one was found external, but close to, the plexus and contained large granular vesicles. The other cell type contained numerous glycogen-like granules, was situated close to or within axon bundles and had processes that extended within and peripheral to nerve bundles as well as being close to smooth muscle cells. Although axon varicosities were opposed to both the processes and cell body of the second type of cell, synaptic-like contacts were not observed.     

GnRH in the spinal subarachnoid space potentiates lordosis behavior in the female rat

Gonadotropin releasing hormone (GnRH) (100 ng) infused directly into the spinal subarachnoid space via a chronically implanted catheter, induced a prompt facilitation of lordosis behavior in estrogen-primed ovariectomized female rats. Facilitation occurred within 5 min and lasted for 3 1/2 hr. This effect of GnRH could be blocked by a GnRH antagonist analog, [D-Phe2, Pro3, D-Phe6] GnRH (250 ng) which also completely abolished lordosis within 2 1/2 hr. The antagonist analog (250 ng) but not a high titre specific anti-GnRH antiserum (enough to neutralize 24 micrograms of GnRH) abolished lordosis when infused alone in the subarachnoid space in estrogen-primed rats. The antagonist but not the anti-GnRH antiserum also significantly suppressed lordosis in estrogen-progesterone primed female rats. Supporting experiments showed that the effects of GnRH and its antagonist may not be due to their effects on supraspinal structures by diffusion from the spinal subarachnoid space. These results may indicate a direct effect of GnRH and its antagonist at the level of the spinal cord and may suggest a possible role for GnRH in the processing of somatosensory information necessary for the trigger of lordosis.     

Direction and linear velocity of flow of cerebrospinal fluid in the spinal subarachnoid space

Summary The direction and velocity of motion of the cerebrospinal fluid in the spinal subarachnoid space was studied in experiments on cats under urethane anesthesia with the aid of radioactive phosphorus (P³²). The latter was introduced into the spinal subarachnoid space at the level of the 6th thoracic vertebra. The activity of cerebrospinal fluid was investigated at definite intervals in the suboccipital and lumbar cisterns and it was revealed that it moved in a candal direction with the velocity of 0.3 mm per min.Diffusion plays a significant part in distribution of radioactive substances in the spinal subarachnoid space.Presented by P. S. Kupalov, Active Member Acad. Med. Sci. USSR     

Ovarian development in athymic nude mice. II. The growth of the oocyte and follicle.

Congenitally athymic mice homozygous for the Mendelian recessive mutation "nude" develop well defined morphological and quantitative changes in the ovarian follicle population. A decline in follicle numbers at 2 months of age is preceded by a retardation in follicle growth at 1 month of age. The growth of the oocyte and its nucleus are not affected by the nude mutation. However, the rate of growth and maximum size of the oocyte nucleolus are reduced in nudes. These developmental events are discussed in relation to the genetic activity of the oocyte, the role of pituitary gonadotrophins in follicular and oocyte growth and the possible role of the thymus gland in these processes.     

An improved method for chronic catheterization of the rat spinal subarachnoid space

A method for chronic catheterization of the rat spinal subarachnoid space is described. The catheters are simple to make and the catheterization procedure circumvents many of the problems associated with earlier published technics.     

神经干细胞移植对小鼠脊髓损伤的影响

目的观察神经干细胞(NSCs)移植对脊髓损伤(SCI)小鼠功能恢复的影响。方法分离、培养、增殖和纯化E14-17d小鼠的NSCs,并通过免疫荧光法对其进行鉴定。将绿色荧光蛋白转基因小鼠的NSCs移植到小鼠脊髓损伤模型体内,术后进行行为学和病理学检测,观察小鼠功能恢复情况。运用改良Allen's法制备小鼠T10-T11脊髓损伤动物模型,动物分为假手术组(12只)、模型组(12只),治疗组(12只)和对照组6(12只)。治疗组每只小鼠自眶静脉注射NSCs悬液200μl(2×10个细胞),对照组只注射DMEM/F12培养基200μl。术后1d、3d、7d、14d、21d、28d和56d,进行BBB运动功能评分和病理学检测,观察植入区细胞生长变化情况。结果 BBB评分显示:治疗组明显高于对照组(<0.01),治疗组与假手术组相比没有明显差异(>0.05),说明NSCs移植后小鼠的行为学得到了明显改善,功能有所恢复。病理学检测发现,移植后NSCs不仅迁移到脊髓损伤区,而且与宿主细胞较好地整合。结论移植的E14-17d胚胎小鼠NSCs不仅可在脊髓损伤部位存活并和宿主细胞整合,而且可促进小鼠后肢运动功能恢复。     

C1型尼曼-匹克症小鼠脊髓胶质细胞的活性变化

目的通过观察C1型尼曼-匹克症小鼠不同脊髓节段星形胶质细胞和小胶质细胞的活性变化,探讨Npc1基因突变对脊髓发育的影响。方法 Npc1~(+/-)小鼠交配繁殖产生Npc~(1-/-)(n=3)和Npc1~(+/+)小鼠(n=3),PCR检测新生小鼠的基因型;选取35日龄的Npc1~(-/-)和Npc1~(+/+)小鼠,采用免疫荧光方法观察对比脊髓不同节段(颈、胸、腰、骶)星形胶质细胞和小胶质细胞的活性变化。采用免疫双染色检测胶质细胞中炎性因子的表达情况,采用免疫印迹方法检测白细胞介素-1β(IL-1β)、SMI31和磷酸化的tau蛋白表达情况。结果在35日龄Npc1~(-/-)小鼠脊髓的各个节段,其背角和腹角的星形胶质细胞和小胶质细胞的活性均明显增强(P0.05),并伴随细胞炎性因子IL-1β表达量的显著增加;同时,脊髓神经丝蛋白和骨架蛋白tau蛋白发生超磷酸化。结论 Npc1基因突变引起脊髓神经胶质细胞发生病理性变化,可能是脊髓神经元病理性损伤的重要原因。     

Effect of levamisole on autologous rosette-forming cells in nude mice.

Levamisole depresses the number of autologous rosette-forming cells (ARFC) in the spleen of nude (congenitally athymic) mice. Intravenous administration of 2.5 mg/kg of levamisole produces maximal depression. This effect appears 15 h after injection and is transient, partially disappearing after 48 hr. Dexamisole is devoid of this depressing activity. Thymopoietin, a thymic hormone, is also shown to lower the level of autologous rosette formation. These results suggest a stereospecific interference of levamisole with the maturation of immature T-cell precursors in a manner resembling the action of thymic hormones.     

Early development of the brain and spinal cord in dysraphic mice

Summary The development and closure of the neural folds was studied in C57BL/6J and loop-tail (Lp) mutant mice by means of scanning electron microscopy on a series of embryos ranging in age from 7.5 to 9.0 days of gestation. The normal embryos (C57BL/6J; +/+; Lp/+) showed a transitional zone of flattened cells lying between the surface ectoderm and neuroepithelial cells at the apices of the neural folds in the presumptive hindbrain and spinal cord, and ruffles occurred at the boundary between the flattened cells and surface ectoderm in regions of the folds which were about to fuse. In the abnormal loop-tail homozygotes (Lp/Lp) which exhibit dysraphism, the ruffles were arranged erratically along the zone of flattened cells. Moreover, at the stage when the folds became apposed and fused in the normal embryos, the abnormals showed ruffles extending the entire length of the unfused folds, thereby distinguishing the abnormals from retarded n normal embryos. Within the neural groove of the hindbrain region, the lateral neuroepithelial cells of the abnormal dysraphic embryos exhibited more flattened surfaces and fewer villous projections than in the normal embryos. The abnormal embryos also lagged behind their normal littermates in converting the body axis from the initial V-shape to the C-shaped configuration.This research was supported by NIH grant no. HD09562 from the National Institute of Child Health and Human Development, USPHS