Antibody-forming capacity of mouse spleen cells after hypoxic hypoxia and the administration of erythropoietin

In CBA mice the absolute and relative (per 10(6) spleen cells) number of antibody-forming cells (AFC) in the spleen was cut by half on the 1st, 4th, and 7th days after acute hypoxia (12 hours, 6700 m), and on the 1st and 4th days after cessation of chronic hypoxia (16 days, 16 hours, 6700 m). The number of AFC in the spleen returned to the normal level on the 7th day after cessation of chronic hypoxia. Single or double erythropoietin injections caused approximately a 1.15--2-fold decrease in spleen AFC number in posthypoxic mice in comparison with control animals.     

Dendritic cells in the rat spleen follicles

Follicles of peripheral lymphoid organs (rat) contain a type of non-lymphoid cell which is capable of arresting antigen-antibody complexes at the cell surface. These so-called dendritic cells can be visualized in immunized rats by staining antigen-antibody complexes with immunohistoperoxidase techniques. The present study concerns a classification of these cells and comparison with known non-lymphoid cell types such as macrophages, marginal metallophils and tingible body macrophages in the rat spleen follicles. Immunoenzyme histochemical and (enzyme) histochemical techniques have been combined in the same tissue sections to correlate the functional capacity of binding immune complexes with morphological characteristics or phagocytic capacity. Dendritic cells show silver affinity but do not demonstrate a characteristic pattern of hydrolytic enzymes or phagocytosis.     

Osteogenic potential of rat spleen stromal cells

Evidence is mounting that an increasing number of cell populations in the adult organism already committed and/or differentiated retain the ability to reprogram themselves and give rise to a different phenotype. Bone marrow stromal cells have long been recognized as early progenitor cells for osteoblasts, chondrocytes, hematopoietic-supportive fibroblasts and adipocytes. Recent reports though have demonstrated a potential of cell populations outside the bone marrow environment to sustain bone formation under specific circumstances. The formation of bone nodules in the spleen of IL-5 transgenic mice has been recently reported (Macias et al. (2001): J. Clin. Invest. 107, 949 - 959). We thus postulated that a cell population exists in the spleen that under particular microenvironmental conditions is able to reprogram itself and pursue a fate other than the tissue-specific one. Therefore we isolated and expanded in vitro spleen-derived stromal cells. After expansion, these cells were challenged with culture conditions designed to induce osteogenic differentiation. We hypothesized that the combination of a proliferating factor (fibroblast growth factor 2) and a differentiating hormone (dexamethasone) would allow us to induce spleen-derived stromal cells to proliferate and at the same time to express osteoblast-specific genes. Thus, spleen-derived stromal cells were isolated from rat spleen and expanded in the presence of fibroblast growth factor 2 and dexamethasone. Once primary cultures reached confluence they were either switched to an osteo-inductive medium or implanted in immunodeficient mice. Although no bone formation was observed in in vivo experiments, in vitro spleen-derived stromal cells were able to deposit a mineralized matrix. Gene expression, as revealed by RT-PCR analysis, evidenced that the deposition of a mineralized matrix was concomitant with the expression of CBFA1 and osteocalcin, along with alkaline phosphatase and bone sialoprotein. Our data suggest that rat spleen-derived stromal cells can undergo osteogenic differentiation in a permissive microenvironment.     

Stereological study of rat spleen following acute ethanol treatment

To investigate the acute effect of ethanol (4 g/kg, i.p.) on spleen adult female Wistar rats were treated intraperitoneally with: a) ethanol (4 g/kg body wt), b) naltrexone (5 mg/kg body wt) followed 45 minutes later by ethanol (4 g/kg body wt) and c) naltrexone (5 mg/kg body wt) alone. Untreated and saline-treated rats were used as controls. Twenty hours after the ethanol treatment the animals were sacrificed and the spleens were removed. A piece of tissue from the central part of each organ was fixed in Bouin's solution. Paraffin sections were stained with hematoxylin-eosin and analysed using stereological measurements. The volume densities of the following tissue compartments: red pulp, white pulp (divided in follicles, periarterioral lymphatic sheath and marginal zone) and the connective tissue were determined. Stereological analysis also included parameters of follicles: the areal numerical density (the number of follicles per 1 mm2 of tissue section), the numerical density (the number of follicles per mm3 of tissue) and the mean follicle diameter. The immunoarchitecture of the spleen was preserved following acute ethanol treatment. Unlike other parameters that were unaffected, ethanol evoked a decrease in both volume density of follicle and the mean follicle diameter. Naltrexone pretreatment had no influence on ethanol-induced changes. The data obtained indicate that a single dose of ethanol has a profound effect on rat spleen affecting the follicles, but the mechanism of its action remains to be elucidated.     

Receptors for IgM on rat spleen cells

Ox red blood cells (ORBCs) sensitized with rat IgM antibodies formed antibody-coated red blood cell rosettes (EAR) around 9% of rat spleen cells freshly prepared at 4 °C, while an enhancement of the rosette-forming cell (RFC) frequency to 18–20% was observed after having exposed the spleen cells to a temperature shift from 4 to 37 °C. Although the temperature shift was found to increase the RFC frequency, a shedding of IgM-Fc receptors IgM-FcR into the medium was also detected (IgM-FcR-I). Regeneration of shed receptors within 5 hr has been proved, and the sheading-regeneration cycle could be repeated several times. IgM-Fc receptors detectable after the temperature shift (IgM-FcR-II) are neither shed nor detectable on freshly prepared spleen cells. This latter type of receptor is expressed only after incubating the cells at 37 °C for an optimal period of 8–10 hr. Both type I and II IgM-FcRs were detected on T lymphocytes as well as B lymphocytes; therefore they do not mark subpopulation. Both receptors are sensitive to trypsin and the activity of both requires Ca²⁺ ions. Shed receptors are able to inhibit EAR formation by cells carrying either IgM-FcR-I or IgM-FcR-II. They are sensitive to higher temperatures and agglutinate ORBCs sensitized by IgM antibodies in the presence of Ca²⁺ ions. EAR-inhibiting capacity was detected in two fractions obtained by gel chromatography of the supernatant after shedding. The elution volume of one of the active fractions corresponds to 130,000 daltons (D), and that of the other to approximately 50,000 D.     

Stimulation of rat spleen cells by staphylococcal enterotoxins

Abstract There is much interest in staphylococcal enterotoxins as T cell mitogens in humans, mice and rabbits. Rat spleen cells were shown to proliferate in response to staphylococcal enterotoxins A and B and toxic shock syndrome toxin-1 at concentrations (5 to 500 ng ml⁻¹) which also stimulate mouse spleen cells. The proliferative response to all these enterotoxins was inhibitted by cyclosporin A, indicating the response to be predominantly that of T cells. These results indicate that the rat provides another convenient model for the analysis of T cell responses to enterotoxins.     

Polyamine content as a marker of radiation injury in the rat spleen

The modifications of the polyamines putrescine (put), spermidine (spd) and spermine (spm) in rat spleen after 3 Gy whole-body irradiation were studied. Rats were irradiated at four different times of the day (00.00, 06.00, 12.00 and 18.00) and sacrificed between 12 h and 62 days after irradiation. Control animals, sacrificed at the same times of the day, showed higher levels of the spd/spm ratio during the hours of light. After irradiation the polyamine content was rapidly and significantly reduced over a period of 20 days. The modification of the amount of spm lasted for a longer period of time. Normal values of polyamine content were reached at later times when the mitotic activity was restored. The results show a close correlation between polyamine concentration and [3H]thymidine uptake.     

Focal regeneration in the rat myocardium following cold injury

Summary The sequential cytological events in the myocardium of the rat were followed for 3 weeks after cold injury by light and electron microscopy. The traumatized area was initially filled with leukocytes and undifferentiated mononucleated cells and subsequently mainly with fibroblasts surrounded by collagen fibers. However, in the margins of the necrotic area repair processes of damaged myocardial cells and probably also the appearance of newly formed cells were evident. The ultrastructural features of these cells were characterized by clusters of ribosomes, numerous mitochondria that were dispersed in the cytoplasm and formation of junctional complexes and transverse tubular systems. Fibrillogenesis was also clearly evident in these cardiomyocytes. The myofibrillar material was initially dispersed in the cytoplasm and associated with clusters of ribosomes and thereafter with presumptive Z-bands and intercalated discs. The myofibrils became further organized in the shape and orientation of those of mature cells two to three weeks after injury. It is concluded that following cold injury regeneration in the mammalian myocardium takes place but is limited to the perinecrotic area. The process resembles the sequential cytological events which occur in cardiomyocytes during embryonic and postnatal development of the ventricular myocardium.     

Characterization of cytoskeletal and neuronal markers in micromass cultures of rat embryonic midbrain cells

Micromass cultures of rat embryonic midbrain cells were characterized with regard to the immunolocalization of neuronal and cytoskeletal markers. Cells taken from gestational day-12 embryos and cultured for 5 days in vitro comprise at least two morphologically distinct cell types:-fibroblast-like cells and neurons. Antibodies to the following markers yieldedpreferential staining of neuronal cells: A2B5 (GQ ganglioside), -aminobutyric acid (GABA), microtubule-associated protein 2 (MAP2), MIPS, neuron-specific enolase (NSE), neural cell adhesion molecule (N-CAM), and tau. Antibodies to -tubulin, -neu, MAPI, and neurofilament (NF-H) stained both neuronal and fibroblast-like cells. Antibodies to glial fibrillary acidicprotein (GFAP) and vimentin failed to immunoreact with any cells in day-5 CNS cultures. SDS-PAGE and Western analysis were employed to determine the specificity of the antibodies and determine the electrophoretic profiles of the markers. We conclude that thepattern of neuronal differentiation in CNS micromass cultures exhibits certain similarities to that observed in vivo. In addition, certain markers identified in this study may be ofpotential utility as (1) biomarkers of chemically-induced developmental neurotoxicity, and (2) indicators of diferential toxicity toward the diverse cell types that comprise the mammalian central nervous system.Portions of these results were presented at the Society of Toxicology Annual Meetings, Seattle, WA, February 1992.     

Engraftment,migration and differentiation of neural stem cells in the rat spinal cord following contusion injury

Background aimsSpinal cord injury is a devastating injury that impacts drastically on the victim's quality of life. Stem cells have been proposed as a therapeutic strategy. Neural stem (NS) cells have been harvested from embryonic mouse forebrain and cultured as adherent cells. These NS cells express markers of neurogenic radial glia.MethodsMouse NS cells expressing green fluorescent protein (GFP) were transplanted into immunosupressed rat spinal cords following moderate contusion injury at T9. Animals were left for 2 and 6 weeks then spinal cords were fixed, cryosectioned and analyzed. Stereologic methods were used to estimate the volume and cellular environment of the lesions. Engraftment, migration and differentiation of NS cells were also examined.ResultsNS cells integrated well into host tissue and appeared to migrate toward the lesion site. They expressed markers of neurons, astrocytes and oligodendrocytes at 2 weeks post-transplantation and markers of neurons and astrocytes at the 6-week time-point. NS cells appeared to have a similar morphologic phenotype to radial glia, in particular at the pial surface.ConclusionsAlthough no functional recovery was observed using the Basso Beattie Bresnahan (BBB) locomotor rating scale, NS cells are a potential cellular therapy for treatment of injured spinal cord. They may be used as delivery vehicles for therapeutic proteins because they show an ability to migrate toward the site of a lesion. They may also be used to replace lost or damaged neurons and oligodendrocytes.     

Tight junction of sinus endothelial cells of the rat spleen

The fine structure of the tight junctions between sinus endothelial cells of the rat spleen and the permeability of such sinus endothelial cells were examined by transmission electron microscopy, using freeze-fracture, triton extraction, and lanthanum-tracer techniques. In freeze-fracture replicas, the segmented strands and grooves of the tight junctions were frequently observed on the basolateral surfaces of the sinus endothelial cells irrespective of the location of the ring fiber. There were one or two wavy-strands or grooves which were, for the most part, oriented parallel to the long cell axis thus forming networks at places. In addition, some strands or grooves were discontinuous while some networks of the junctional strands were not closed. These strands also occasionally lacked intramembranous particles in the tight junctions. The junctional strands run apicobasically at certain sites. In the vertical sections of the sinus endothelial cells treated with lanthanum nitrate, although no tight junctions were observed wherever the endothelial cells were apposed, most of them were situated on the basal part of the lateral surfaces of the adjacent endothelial cells. Several fusions of the junctional membranes were observed in a vertical section of the lateral surfaces of the adjacent endothelial cells. The intercellular spaces of the adjacent endothelial cells except for the fusion of the junctional membranes, were electron dense and the infiltration of lanthanum nitrate was found not to be interrupted by these tight junctions. Based on these observations, the molecular 'fence' and paracellular 'gate' functions of the tight junctions in the sinus endothelial cells are discussed.     

The surface-connected canalicular system in the sinus endothelial cells of rat spleen

The existence of a surface-connected canalicular system in the splenic sinus endothelial cells of the rat has been demonstrated by transmission electron microscopy with lanthanum nitrate acting as a tracer for the extracellular space. In addition, the three-dimensional arrangement of the canaliculi has been revealed by computer-aided reconstruction. The surface-connected canalicular system of the sinus endothelial cells consists of slender canaliculi that are branched, anastomosed, and that show continuity with the plasma membrane. They twist in and out among the organelles and are often found in close apposition to the spherical invaginations of the plasma membrane and run alongside them. Canaliculi which are not infiltrated by lanthanum nitrate take the form of electron-lucent tubules and are accompanied by numerous spherical invaginations of the plasma membrane. From a computer-aided reconstruction, the canaliculi, which invaginate from various sites of the plasma membrane, have been found to be continuous with each other and to penetrate to the surface of the sinus endothelial cell; they also branch and anastomose to form a complex network in the cytoplasm. Although the surface-connected canalicular system in blood platelets and thrombocytes is believed to function as the main route for the discharge of granules and the uptake of foreign materials and also to take part in the storage and transport of calcium, it is unclear at present whether the network of the surface-connected canalicular system in splenic sinus endothelial cells has any physiological significance.