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PubMed1983

A new recycling technique for human placental cotyledon perfusion: application to studies of the fetomaternal transfer of glucose, inulin, and antipyrine.

A previously described technique has been modified to permit the continuously recirculating perfusion of the separate maternal and fetal circulations of an isolated cotyledon of human placenta. Viability of the perfused cotyledons was established by measurements of oxygen consumption (average, 0.18 ml/gm/hr), glucose utilization (average, 1.0 mg/gm/hr), and lactate production (less than 0.01 mumol/gm/hr), and integrity of the placental barrier by the failure of India ink, 125I-albumin, or 35S-sulfobromophthalein to cross from fetal to maternal circulation. Clearance of 3H-inulin from the fetal circuit, 0.0059 +/- 0.0005 (SE) ml/min/gm, corresponded to 2.5% of its clearance by the adult human kidney. Clearance of 14C-antipyrine was 0.013 +/- 0.003 ml/min/gm. After introduction into the fetal circuit, the observed appearance of both inulin and antipyrine in the maternal circuit closely paralleled curves predicted by a simple mathematical model. The use of a continuously recirculating perfusion system is technically feasible, and has advantages over the single-pass technique for studying transplacental transfer of metabolites with a low efficiency of extraction.

Animals↗
PubMed1985

Protein blotting: detection of proteins with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes.

Methods to detect "native" proteins immobilized on nitrocellulose membranes in spot tests or on blots prepared from polyacrylamide slab gels after electrophoretic separation are described. Gold sols were found to be useful as general stains for proteins: They are polychromatic, yield an indelible record, and are complementary to india ink as protein stains because these two stains have different sensitivities for a number of proteins tested. For detection of wheat germ lectin (WGL)-binding glycoproteins, avidin-peroxidase was an effective enzyme probe, because the glycoportion of the avidin moiety possesses binding affinity to WGL. Glycocomponents in human parotid saliva were detected with this probe and with the following biotin-conjugated lectins as intermediary probes: soybean lectin, Bandeiraea simplicifolia lectin, Lotus tetragonolobus lectin, and kidney bean lectin. Autoclaving blots prior to probing eliminated endogenous peroxidase activity. Concanavalin A and WGL were separated by isoelectric focusing and detected on blots with horseradish peroxidase and avidin-peroxidase, respectively. The versatility of the biotin/avidin system was used to detect other lectins on similar blots using biotin-conjugated glycoproteins as intermediary probes: Helix pomatia lectin and B. simplicifolia lectin were detected with biotinyl neoglycoproteins, and kidney bean lectin with biotin-conjugated components of parotid saliva.

Biotin↗
PubMed1988

The use of polyvinylidenedifluoride membranes as a general blotting matrix.

A fast chemical staining of protein by Coomassie blue on a stable nonshrinking membrane of polyvinylidenedifluoride is described. The procedure is especially useful for detection of nonlabeled marker proteins in comparison to specifically detected proteins. The membrane is highly suitable for other chemical stains like silver, gold, or india ink for immunostaining, lectin binding, and binding of radioactive ligands like 45Ca. Blocking of the background against nonspecific binding to proteins was studied using different reagents. A multiple slit plate is described for volume-saving simultaneous application of multiple reactants on one protein-loaded membrane.

Glycoproteins↗
PubMed1988

Protein blotting with direct blotting electrophoresis.

Direct blotting electrophoresis, a method designed to be of general application for the separation and electroblotting of macromolecules, has been adapted to produce protein blots suitable for subsequent processing by standard techniques such as dye staining or immunological detection. After their separation in a very short gel the protein bands are electrophoresed out of the gel onto an immobilizing matrix. The matrix which is moved across the bottom of the gel by a conveyor belt binds these proteins with high affinity. Once the protein samples have been loaded onto the gel and electrophoresis has been started, no further intervention is needed until the blot is completed. The total expenditure of time for such a direct blot is less than 4 h for a mixture of proteins in the molecular weight range of 14-70 kDa. The staining sensitivity of directly blotted proteins is about 200 ng protein per band as revealed by India ink staining.

Collodion↗
PubMed1991

Detection of plant virus coat proteins on whole leaf blots.

Coat proteins of several different plant viruses were transferred from entire leaves of infected hosts to nitrocellulose membranes in 2 min using a hydraulic laboratory press. Protein transfer was even across the leaf as evidenced by India ink staining of blots. Coat proteins of four different viruses were detected using virus specific IgG. The presence of viral coat proteins was observed as small intense spots whose distribution was irregular and coincided most frequently with the presence of chlorosis on the leaves. Detection was sensitive, and accurately reflected the distribution of virus symptoms across leaves.

Capsid↗
PubMed1983

Retrograde access of antigens to the minor salivary glands in the monkey Macaca fascicularis.

Several authors have postulated that the ducts of minor salivary glands may provide pathways for the presentation of oral antigens to cells involved in a local immune response. This would imply a retrograde passage of oral antigens into the minor salivary glands. Various sites of the oral mucosa of anaesthesized M. fascicularis monkeys were labelled topically with a multiple tracer consisting of horseradish peroxidase (HRP), ferritin and Indian ink for a continuous period of 4-5 h. Serial cryosectioning of labelled lips and soft palates revealed retrograde passage of this tracer deep into some of the corresponding minor salivary glands. Minor salivary gland ducts can therefore serve as "portal of entry" for oral antigens, at least in the M. fascicularis monkey.

Animals↗
PubMed1987

The development of mandibular molar tooth germs isografted in the mouse spleen.

Tooth germs taken from 13-day-old embryonic mice and isografted in the spleen of adult mice were examined by light and electron microscopy. Well-organized tooth structures from the early cap stage to fully developed and mineralized mature teeth were obtained up to day 60 after transplantation. Germs on day 2 were similar to those prior to the onset of grafting but reached the late cap stage of development on day 4. On day 6, enamel and dentine formation were initiated and inner enamel epithelium and dental papilla cells were polarized. On days 10-15, enamel-matrix secretion was completed and almost all ameloblasts had become resorptive enamel epithelium. India ink injected from the recipient caudal vein accumulated to the capillaries within the pulp throughout the newly-formed vessels. On day 20, defined root formation had begun but occasionally irregular and cellular osteodentine was formed in root areas. On day 30, transplants were covered with reduced enamel epithelium and acellular cementum was formed at the root areas together with rudimentary periodontal ligament fibres. Cellular cementum became thicker up to day 40. There was little evidence of cellular infiltration from recipient tissue up to day 60. The spleen seems to be a suitable site for transplantation of tooth germs.

Animals↗
PubMed1995

The angioarchitecture of the rat mandibular joint synovium.

The synovium (or synovial membrane) lines all intra-articular surfaces of synovial joints except for the articulating surfaces. This study describes the angioarchitecture of the synovium of the adult rat temporomandibular joint using microvascular corrosion casts and scanning electron microscopy. To corroborate findings, India ink-injected, thick-sectioned specimens (200 microns) and haematoxylin and eosin-stained tissue sections (7 microns) were analysed. Rostrally, the synovial membrane was fed by several layers of vessels branching towards the superficial lining of highly convoluted capillary loops. Arterioles were rare, and venules organized in large plexuses. The membrane thinned out in the caudal direction. Villi were present on the surface. They were flat, with a core of two parallel linear feeder vessels, one arteriole and one venule, which were interconnected by capillary loops. At the periphery of the articulating surfaces, the vessels of the synovial layer were flatter. Caudally, the synovial membrane of the richly vascularized retrodiscal pad showed many synovial folds. Feeder vessels were directed rostrally and branched in all directions. The number of subdivisions depended on the size of the synovial fold. Again, venules were organized in plexuses and lining capillaries were convoluted. The densely capillarized synovial membrane with its folds and villi is considered well adapted to serve synovial fluid production and joint dynamics.

Animals↗
PubMed1995

The angioarchitecture of the rat mandibular joint bilaminar zone.

In mammals, temporomandibular-joint articular disc attachments have a bilaminar pattern. The three-dimensional angioarchitecture of the bilaminar zone in the adult rat was described using scanning election microscopy of microvascular corrosion casts (47 specimens) and light microscopy of Indian ink-injected, cleared, thick-sectioned temporomandibular joints (6 specimens). The bilaminar zone had an axial core of feeder vessels composed primarily of flat venules which were organized in plexuses. Arterioles were few and slender. In both laminae, there were usually three branching levels until vessels approached the surface of the lamina, where a dense capillary meshwork was formed. Both laminae ended abruptly at the periphery of the avascular disc with a single, slightly undulating marginal vessel. This marginal vessel, which faced the avascular disc, was definitely larger in diameter than the other superficial capillaries and was rather a postcapillary venule than a capillary. Functionally, this marginal venule might be important in sustaining nutrition of the avascular disc centre by allowing bidirectional blood flow during jaw movements.

Animals↗
PubMed1996

Preparation and properties of sterically stabilized hexadecylphosphocholine (miltefosine)-liposomes and influence of this modification on macrophage activation.

The aim of the study was to investigate for the first time the preparation, physical properties and macrophage activating effect of sterically stabilized liposomes made from hexadecylphosphocholine (HPC, Miltefosine) using different poly(ethylene glycol) lipids for coating. We could demonstrate that it is possible to prepare different liposomal vesicle types (MLV, SUV and LUVET) without any problem and with a high stability in buffer (release of hydrophilic marker was < 5% after half a year) and in plasma (t1/2 up to several days). The preparation method, including size of polycarbon membrane filter used for the preparation of LUVETs had the main influence on vesicle size and size distribution. The addition of a charged lipid like DCP and different amounts of PEG-lipid up to 10% had no effect on size and stability of PEG-LUVETs. A comparison of activating potency of PEG-HPC-vesicles with commonly used HPC-liposomes was performed with mouse peritoneal macrophages. HPC-liposomes induced a clear release of NO and TNF from mouse peritoneal macrophages especially in a synergistical action with LPS. On the contrary the effect of PEG-liposomes was similar to control cells after a combined activation in vivo/in vitro. The reduced interaction of these liposomes with the MPS was also demonstrated by an unchanged carbon ink uptake after treatment of mice (i.p.) with liposomes prepared with and without PEG-lipid. PEG-HPC-liposomes combine the advantages of HPC, liposomes and PEG-coating, resulting in a promising preparation for treatment of mammary cancers.

Animals↗
PubMed1980

Identification of neurons contributing to presynaptic inhibition in Aplysia californica.

Electrical stimulation of the connectives presynaptically inhibits the PSP from cell L10 to the left upper quadrant cells (LUQC). The present report describes the properties of some of the individual neurons contributing to this response. Action potentials produced in a cluster of cells in the abdominal ganglion reduce the amplitude of the L10-LUQC PSP for periods greater than 30 sec. At least some of their inhibitory action is mediated by a slow hyperpolarization of L10 which results in a decreased transmitter release. In other cases, however, the inhibition is produced with no significant alteration of L10 membrane potential, indicating that additional mechanisms may also be present. The neurons producing these effects are approximately 75 microns in diameter and are located on the left ventral surface of the ganglion. They have axons in the connectives and are thus activated by stimuli previously utilized to produce presynaptic inhibition. They appear to be some of the same cells that produce a slow inhibition of ink motoneuron L14; one of these has been identified as L32. The identification of these cells allows for the further biochemical, biophysical and morphological analysis of the events underlying presynaptic inhibition.

Animals↗
PubMed1982

Meningeal relations of the rat hypothalamo-hypophyseal system. Extravascular fluid spaces in and around the median eminence.

Meningeal relations of the rat pituitary and basal hypothalamus including the pituitary stalk and median eminence, were studied by both light and electron microscopy, and by ink perfusion techniques. The dura mater encapsulates the pituitary gland. The arachnoid covers the basal surface of the hypothalamus except the caudal two-thirds of the median eminence and the pituitary stalk. No arachnoidea, i.e. no subarachnoideal space, was found in the sella. Pia mater covers the entire basal surface of the hypothalamus, including the median eminence and the pituitary stalk, but encapsulates only the posterior lobe of the pituitary. The superficial portal vessels (primary plexus and portal veins) are located in the subdural space, covered by the pia mater. The deep vessels in the median eminence are surrounded by the perivascular space, which is bordered by the vascular and neuropil basement membranes, and separated from the external fluid space around the median eminence only by the pia mater. This special topography suggests that molecules present in the portal vessels or in the cerebrospinal fluid, or in and around the pituitary gland, readily move from one liquid space to another.

Animals↗
PubMed1986

Immunohistochemical investigation of regional cerebral ischemia in the gerbil: occlusion of the posterior communicating artery.

Evolution, progression and recovery of neural damage during and following cerebral ischemia were investigated in the gerbil after occlusion of a posterior communicating artery and by using the immunohistochemical reaction for tubulin and creatine kinase BB-isoenzyme which are enriched in the neuronal structure and the reaction for astroprotein which is specific for astrocytes. The transcardiac perfusion study with India ink revealed marked hypoperfusion diffusely in the hippocampus and moderately in the thalamus on the occluded side. The earliest immunohistochemical lesion, manifested as loss of the reaction for tubulin and creatine kinase BB-isoenzyme in dendrites and nerve cell bodies, was found in the CA1 and CA2 region of the hippocampus after ischemia for 4 min, while it took 10 min before the earliest lesion became visible in the ventral nucleus of the thalamus and it took over 1 h before scattered lesions evolved in granular cells of the dentate gyrus. The staining with hematoxylin-eosin was much less sensitive in detection of early ischemic lesions. After re-establishment of blood flow to the posterior communicating artery, the ischemic lesions which were visualized with the reaction for tubulin or creatine kinase BB-isoenzyme disappeared or reduced the size, if the ischemic period was brief. Beyond a certain ischemic period, the lesion expanded further during the early postischemic period. The reaction for astroprotein visualized reactive astrocytes even in the area without any abnormalities with other reactions, an evidence of subtle ischemic insults.

Animals↗
PubMed1989

Mosaic reconstruction of blood vessels in mouse neocortical tissue transplanted into the third ventricle of rat brain.

Newborn mouse neocortical tissue was transplanted into the third ventricle of rat brain and the reconstruction and the origin of the blood vessels were investigated by using a monoclonal antibody against mouse endothelial surface antigen-1 (MESA-1). It was clearly demonstrated that some of the blood vessels in the graft originated in the donor mouse neocortical tissue. An India ink perfusion experiment revealed that the blood was supplied to the MESA-1-positive blood vessels. Furthermore, electron microscopic immunohistochemical studies demonstrated the existence of a mosaic reconstruction of blood vessels which consisted of mouse- and rat-derived vascular endothelial cells. It was concluded that the blood vessels originating in the donor tissue and those originating in the host tissue inoculate with each other in the grafted tissue.

Animals↗
PubMed1990

Guanethidine-induced adrenergic sympathectomy augments endoneurial perfusion and lowers endoneurial microvascular resistance.

Chronic administration of guanethidine sulfate in the rat induces a selective adrenergic neuropathy. We studied the effects of guanethidine-induced adrenergic sympathectomy on rat sciatic nerve blood flow (NBF), microvascular resistance (MR), vessel caliber and norepinephrine (NE) content. A control group of animals was studied following chronic administration of mammalian Ringer's solution. NBF and MR were measured with an endoneurial microelectrode, using the technique of hydrogen clearance (HC). Following HC, the sciatic nerve was perfused with India Ink, removed, frozen and sectioned. Measurements were made of endoneurial microvessel numbers, diameter, circumference and area. The contralateral sciatic nerve was removed for measurements of NE content. In guanethidine-treated animals we observed elevated NBF, reduced MR and dilated microvessels. Numbers of microvessels and fascicular areas were similar to controls. NE content was markedly reduced following sympathectomy. These studies suggest that NBF, unlike cerebral blood flow (CBF), is regulated by its adrenergic input. Removal of adrenergic innervation of the vasa nervorum appears to result in a loss of tonic vasoconstrictive action.

Animals↗
PubMed1991

Distribution of extracellular tracers in perivascular spaces of the rat brain.

Large molecular weight tracers (india ink or albumin labeled with colloidal gold, Evans blue or rhodamine) were micro-injected into the perivascular space of an artery or vein on the brain surface, or within the cerebral cortex or the subarachnoid space of anesthetized rats. The subsequent distribution was followed both under intravital microscopy, in order to outline the pathways and direction of tracer movement, and in histological section, in order to describe the pathways of flow at the light and electron microscopic level. The tracers remained largely in the perivascular spaces and in the interconnecting network of extracellular channels, including the subpial space and the core of subarachnoid trabeculae. Tracer also leaked across the pia into subarachnoid CSF. Bulk flow of fluid within the perivascular space, around both arteries and veins, was suggested from video-densitometric measurements of fluorescently labeled albumin. However, this flow was slow, and its direction varied in an unpredictable way. These results confirm that perivascular spaces may serve as channels for fluid exchange between brain and CSF, but do not support the idea that CSF circulates rapidly through brain tissue via perivascular spaces.

Animals↗
PubMed1984

The vascular territory of the acromiothoracic axis.

The precise vascular territory and the variations of the acromio-thoracic axis were investigated in a series of 60 fresh cadavers and 50 formalin fixed specimens using dissection, ink injection and barium radiographic studies. The sternocostal portion and the clavicular head of the pectoralis major were found to have virtually independent vascular and nerve supplies. The pectoral artery supplied the former, whereas the deltoid artery nourished the latter. The dominant supply from the pectoral artery to the rib cage was found to enter around the fourth rib in the mid clavicular line. This supply is associated with a previously undescribed origin of the pectoralis major muscle in this region. The supply to the sternum was determined as indirect via the captured territory of the internal mammary system. The dominant supply to the skin from the pectoral artery arose laterally along the free lower border of the muscle as fasciocutaneous branches. The deltoid artery supplies the skin over the shoulder by numerous small branches which emerge from the intramuscular septa of the deltoid muscle. In addition a large axial artery was noted. In most cases this arose from the deltoid artery or its acromial branch and coursed laterally. It is noteworthy that the majority of skin paddles of the pectoralis major myocutaneous flap currently used in clinical practice are designed medially and inferiorly around the perimeter of the muscle and onto the rectus sheath. In these situations such flaps are not supplied directly by the pectoral artery. In fact, they are supplied indirectly by cutaneous branches belonging to the internal mammary/superior epigastric system which are captured by arterial connections with the pectoral artery. These occur predominantly in the pectoralis major muscle. Suggestions, based on these anatomical studies, are offered to improve the versatility and safety of flaps designed in this area.

Acromion↗