Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice.

This study sought to determine whether angiogenic blood vessels in disease models preferentially bind and internalize cationic liposomes injected intravenously. Angiogenesis was examined in pancreatic islet cell tumors of RIP-Tag2 transgenic mice and chronic airway inflammation in Mycoplasma pulmonis-infected C3H/HeNCr mice. For comparison, physiological angiogenesis was examined in normal mouse ovaries. We found that endothelial cells in all models avidly bound and internalized fluorescently labeled cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]/cholesterol or dimethyldioctadecyl ammonium bromide [DDAB]/cholesterol) or liposome-DNA complexes. Confocal microscopic measurements showed that angiogenic endothelial cells averaged 15-33-fold more uptake than corresponding normal endothelial cells. Cationic liposome-DNA complexes were also avidly taken up, but anionic, neutral, or sterically stabilized neutral liposomes were not. Electron microscopic analysis showed that 32% of gold-labeled liposomes associated with tumor endothelial cells were adherent to the luminal surface, 53% were internalized into endosomes and multivesicular bodies, and 15% were extravascular 20 min after injection. Our findings indicate that angiogenic endothelial cells in these models avidly bind and internalize cationic liposomes and liposome-DNA complexes but not other types of liposomes. This preferential uptake raises the possibility of using cationic liposomes to target diagnostic or therapeutic agents selectively to angiogenic blood vessels in tumors and sites of chronic inflammation.

Innervation of the guinea pig trachea: a quantitative morphological study of intrinsic neurons and extrinsic nerves.

The innervation of the guinea pig trachea was studied in wholemount preparations stained for acetylcholinesterase, catecholamines, and substance P immunoreactivity and by electron microscopy. The majority of parasympathetic and afferent nerve fibres arrive from the vagus via branches of the recurrent laryngeal nerves. The recurrent laryngeal nerves are composed of several fascicles comprising 600-700 small myelinated fibres (2-5 microns diameter) and about 1,000-2,000 unmyelinated fibres; both components exit from the nerve and project in fine branches to the trachea. A separate component of 200-250 large myelinated fibres (more than 5 microns diameter) runs the full length of the nerve and innervates the striated muscles of the larynx. The recurrent laryngeal nerves are slightly asymmetric in their origin, length, number, and composition of fibres, with the right nerve being shorter but with more numerous and thinner myelinated fibres. At the distal end of the recurrent nerve, a fine branch called the ramus anastomoticus connects it to the superior laryngeal nerve. In the tracheal plexus, there are on average 222 ganglion cells (range 166-327), distributed mostly in small ganglia of 12 or fewer neurons. The ganglionated plexus is situated entirely outside the tracheal wall, overlying the smooth muscle. Ligation experiments show that sympathetic nerve fibres reach the trachea with the recurrent nerves via anastomoses between the sympathetic chain and vagus nerves, or occasionally with recurrent nerves directly, the largest being at the level of the ansa subclavia. There are also perivascular sympathetic nerve plexuses. Substance P immunoreactive fibres enter the trachea from the vagus nerves and by pathways similar to those of sympathetic nerves. There are also paraganglion cells within the recurrent laryngeal nerve that contain catecholamines and are surrounded by substance P immunoreactive fibres. After cervical vagotomy, all the large myelinated fibres of the ipsilateral recurrent laryngeal nerve degenerate and so do all but 10 or 20 small myelinated fibres and all but a few unmyelinated fibres. Degenerating fibres are found within the entire tracheal plexus, indicating bilateral innervation. The small myelinated fibres that survive cervical vagotomy probably represent sympathetic or afferent nerves with their cell bodies located in sympathetic or dorsal root ganglia.

Substance P-immunoreactive sensory axons in the rat respiratory tract: a quantitative study of their distribution and role in neurogenic inflammation.

Substance P is one of the peptides released from sensory nerves that mediate "neurogenic inflammation." Although substance P-immunoreactive (SP-IR) axons are known to be present within the mucosa of the respiratory tract, the relative extent of the innervation of various components of the mucosa is not known. Therefore, we determined the distribution and number of SP-IR axons in the rat trachea and bronchi, by using immunohistochemistry on tissue whole mounts. Specifically, we sought to learn whether these axons directly innervate the postcapillary venules involved in neurogenic plasma extravasation, the arterioles involved in neurogenic vasodilatation, and the airway smooth muscle involved in bronchoconstriction in pathogen-free, adult male F344 rats. We found that 90% of the SP-IR axons were single axons, usually having varicosities. Eighty-five percent of these were in the epithelium, 6% innervated arterioles, and the remainder elsewhere in the lamina propria. Only 10% of the mediator-sensitive postcapillary venules (i.e., venules labeled with Monastral blue pigment after challenge with capsaicin or substance P) were within 10 microns of SP-IR axons. SP-IR axons were more than 10 times as frequent in the smooth muscle of the distal bronchi as in the trachea. Capsaicin pretreatment (168 mg/kg over 7 days) reduced the number of SP-IR axons in the trachea by 96%, which is consistent with their being sensory. Unilateral vagotomy reduced the number of SP-IR axons bilaterally in the trachea and ipsilaterally in the main stem bronchus. Using an antibody to Protein Gene Product 9.5 as a nonspecific marker for all nerves in the trachea, we determined that SP-IR axons constituted 90% of the axons in the epithelium, 32% of the axons on arterioles, and only 4% of the axons in the smooth muscle. We conclude that most SP-IR nerves in the trachea are sensory axons and most of these axons end in the epithelium. SP-IR axons innervate mucosal arterioles, but few innervate postcapillary venules. Therefore, the mechanism by which sensory axons evoke plasma extravasation from these venules is likely to involve the diffusion of the peptide or a secondary mediator from the epithelium or from the arterioles upstream.

An ultrastructural study of nerve profiles in the myenteric plexus of the rabbit colon.

The ultrastructure of the myenteric plexus from the rabbit colon was examined in both conventionally fixed tissue and also material fixed with the chromaffin method. Montages of the ganglia were analysed semi-quantitatively. Six main types of axon profile are described and classified on a morphological consideration of the vesicle population. Most axon types formed synapses with myenteric neurons. Two kinds of chromaffin-positive nerve fibre were seen, one containing a predominance of small granular vesicles, the other containing many flattened vesicles. The difficulties in relating axon profile types to putative transmitters are discussed.

An ultrastructural study of neurons and non-neuronal cells in the myenteric plexus of the rabbit colon.

The myenteric plexus of the rabbit colon showed a degree of structural organization that was unusually high for the peripheral nervous system, providing a basis for the complex integrative activity which is known to occur. It resembled central nervous tissue in several respects: a wide range of neuron types was present; the proportion of glial cells to neurons was about 2:1; and there was a densely packed, avascular neuropil, not penetrated by connective tissue. Most neurons had at least one surface exposed to the extraganglionic space. Clear evidence was obtained for spontaneous neuronal degeneration. Three types of non-neuronal (glial) cells were observed: Type 1, which was most common, contained many 10 nm 'gliofilaments' and resembled enteric glial cells or astrocytes in the central nervous system; Type 2, composing about 5% of the glial cells, had few filaments; Type 3 was seen only rarely, had a small dark nucleus, little cytoplasm, may have been of extraganglionic origin and resembled microglia of the central nervous system. Fibroblast-like cells were also present in extraganglionic sites. Schwann cells could not be identified within the myenteric ganglia.

Endocytosis and recycling of neurokinin 1 receptors in enteric neurons.

Neurotransmission depends on the availability of transmitter and on the presence of functional, high-affinity receptors at the plasma membrane that are capable of binding ligand. The pathway, mechanism and function of endocytosis and recycling of the substance P or neurokinin 1 receptor in enteric neurons were studied using fluorescent substance P, receptor antibodies and confocal microscopy. In both the soma and neurites, substance P induced rapid, clathrin-mediated internalization of the neurokinin 1 receptor into early endosomes, which also contained the transferrin receptor. After 4-8 h, there was a return in surface neurokinin 1 receptor immunoreactivity in the soma, which was not prevented by cycloheximide, and was thus independent of new protein synthesis. This return was prevented by acidotropic agents, therefore required endosomal acidification. This suggests that the neurokinin 1 receptor recycles in the soma. In contrast, in neurites, substance P and the neurokinin 1 receptor remained in endosomes and recycling was not detected. Neurons of the myenteric plexus were heavily innervated by substance P-containing nerve fibers, and K(+)-stimulated release of endogenous substance P from cultured neurons induced internalization of the neurokinin 1-receptor. Therefore, endogenous substance P may induce endocytosis of the neurokinin 1 receptor. In the soma, endocytosis and recycling correlated with loss and recovery of functional binding sites for substance P. suggesting that this process contributes to the regulation of peptidergic neurotransmission. Thus, ligand-induced endocytosis of the neurokinin 1 receptor in myenteric neurons is associated with a loss of surface receptors and functional binding sites. Since release of endogenous substance P induces neurokinin 1 receptor internalization, and neurokinin 1 receptor neurons are innervated by substance P-containing fibers, endocytosis of neuropeptide receptors may regulate neurotransmission.

Neurogenic plasma leakage in mouse airways.

1. This study sought to determine whether neurogenic inflammation occurs in the airways by examining the effects of capsaicin or substance P on microvascular plasma leakage in the trachea and lungs of male pathogen-free C57BL/6 mice. 2. Single bolus intravenous injections of capsaicin (0.5 and 1 micromol kg(-1), i.v.) or substance P (1, 10 and 37 nmol kg(-10, i.v.) failed to induce significant leakage in the trachea, assessed as extravasation of Evans blue dye, but did induce leakage in the urinary bladder and skin. 3. Pretreatment with captopril (2.5 mg kg(-1), i.v.), a selective inhibitor of angiotensin converting enzyme (ACE), either alone or in combination with phosphoramidon (2.5 mg kg(-1), i.v.), a selective inhibitor of neutral endopeptidase (NEP), increased baseline leakage of Evans blue in the absence of any exogenous inflammatory mediator. The increase was reversed by the bradykinin B2 receptor antagonist Hoe 140 (0.1 mg kg(-1), i.v.). 4. After pretreatment with phosphoramidon and captopril, capsaicin increased the Evans blue leakage above the baseline in the trachea, but not in the lung. This increase was reversed by the tachykinin (NK1) receptor antagonist SR 140333 (0.7 mg kg(-1), i.v.), but not by the NK2 receptor antagonist SR 48968 (1 mg kg(-1), i.v.). 5. Experiments using Monastral blue pigment as a tracer localized the leakage to postcapillary venules in the trachea and intrapulmonary bronchi, although the labelled vessels were less numerous in mice than in comparably treated rats. Blood vessels of the pulmonary circulation were not labelled. 6. We conclude that neurogenic inflammation can occur in airways of pathogen-free mice, but only after the inhibition of enzymes that normally degrade inflammatory peptides. Neurogenic inflammation does not involve the pulmonary microvasculature.

Plasma extravasation induced in rat trachea by 6-OHDA is mediated by sensory nerves, not by sympathetic nerves.

6-Hydroxydopamine (6-OHDA) stimulates the release of catecholamines from sympathetic nerves. This stimulation has been proposed as the basis of the 6-OHDA-induced increase in vascular permeability in the rat knee joint. We sought to determine whether 6-OHDA increases vascular permeability in the rat trachea through a similar mechanism. We also sought to determine whether sympathetic nerves contribute to the plasma extravasation caused by stimulating sensory nerves with capsaicin. In anesthetized rats, an intratracheal infusion of 6-OHDA caused more Monastral blue extravasation than did an infusion of vehicle (area density, 23 +/- 3% vs. 9 +/- 1%). Chemical sympathectomy, which reduced the number of tyrosine hydroxylase-immunoreactive nerves by 98%, did not reduce the amount of extravasation induced by 6-OHDA. However, pretreatment with capsaicin, which reduced the number of substance P-immunoreactive nerves by 95%, reduced the Monastral blue extravasation induced by 6-OHDA by 98%. Extravasation induced by stimulating sensory nerves with capsaicin was not significantly different in tracheae with or without sympathetic nerves. We conclude that in the rat trachea infusion of 6-OHDA causes plasma extravasation by stimulating sensory nerves, not by stimulating sympathetic nerves. Furthermore, sympathetic nerves are not essential for the plasma extravasation induced by capsaicin.

Glial cells in the guinea pig myenteric plexus are dye coupled.

Glial cells in the myenteric plexus of the guinea pig small intestine were stained intracellularly with Lucifer yellow and horseradish peroxidase. The cells were identified by both their electrophysiological characteristics and by their morphology. Injection of Lucifer yellow, which is known to cross gap junctions, resulted in the staining of many (up to about 100) glial cells. The staining pattern was comparable to the immunostaining of glia with an antiserum for S-100 protein. In contrast to Lucifer yellow, horseradish peroxidase (which does not cross these junctions), was confined to the injected cell. It is concluded that enteric glia are coupled, presumably by gap junctions. This finding indicates that in addition to structural and biochemical similarities, enteric glia may share certain physiological characteristics with central nervous system astrocytes.

The enteric nervous system in tissue culture. II. Ultrastructural studies of cell types and their relationships.

Tissue culture preparations of the myenteric plexus from the guinea pig taenia coli have been studied by electron microscopy. Three main cell types can be identified: neurons, enteric glial cells and fibroblasts. The ultrastructure of these cells resembles that of the same cells in situ. Neuronal processes form close associations with other neurons and glial cells, but not with fibroblasts. After extended periods in culture, neurons and glial cells form aggregates of cells which resemble ganglia of the myenteric plexus in situ, having a compact neuropil and synapses between neuronal elements. Aggregates are connected to each other by thick bundles of neurites. Vesicle-containing nerve profiles are common; the majority contain a predominance of small agranular vesicles, but some contain many large granular or large opaque vesicles; profiles may also contain variable mixtures of these kinds of vesicles.

The enteric nervous system in tissue culture. III. Studies on neuronal survival and the retention of biochemical and morphological differentiation.

The maintenance of differentiated properties and survival rates of enteric neurons, grown in explant cultures for periods of up to 3 weeks, was studied. Using catecholamine fluorescence, immunohistochemistry and autoradiography, it was found that adrenergic neurons, VIP-containing neurons and putative GABAergic neurons, which constitute small subpopulations of guinea pig myenteric neurons in vivo, were all represented in plexus explants after maintenance in culture for 2-3 weeks. The pattern of expression of the transmitter-related enzymes, acetylcholinesterase and monoamine oxidase, paralleled that found in in situ studies. Investigation of neuronal structure by intracellular injection of horseradish peroxidase revealed that the cultured neurons continue to express the wide diversity in gross morphology which characterizes these cells in vivo. Employing autoradiography following uptake of [3H]GABA to label putative GABAergic neurons, their survival rate from days 1 to 15 of culturing was determined. No neuronal death was detected between days 1 and 5, while the number of neurons decreased between days 5 and 15. These observations suggest that enteric neurons maintained in explant cultures survive well and maintain to a high degree their histochemical and morphological properties.

Direct visualization by scanning electron microscopy of the preganglionic innervation and synapses on the true surfaces of neurons in the frog heart.

A simple and flexible technique is reported for scanning electron microscopic observation of the true surfaces of neurons, and the distribution of glial cells, preganglionic nerve fibers and synapses on them. The interatrial septum of the frog was chosen as a convenient preparation, and connective tissue and glial cells were successively removed by incubation in collagenase and protease solutions. The three-dimensional relationships between presynaptic fibers and target neurons can be observed with high resolution over long distances. Some neurons have multiple innervation. Varicosities can be seen clearly, and where their interior has been exposed, synaptic vesicles can be discerned.

Tracheal parasympathetic neurons of rat, mouse and guinea pig: partial expression of noradrenergic phenotype and lack of innervation from noradrenergic nerve fibres.

Noradrenergic nerves were studied in whole-mount preparations of the rat, mouse and guinea pig trachea by means of glyoxylic acid-induced catecholamine fluorescence and dopamine beta-hydroxylase immunoreactivity. In an effort to raise tissue levels of catecholamines, some specimens were also treated with the monoamine oxidase inhibitor pargyline, and with L-DOPA, a precursor of noradrenaline. Noradrenergic nerve fibres were detected around blood vessels, within the tracheal smooth muscle and in the mucosa, but never around or in the proximity of neurons of the tracheal ganglia, even after amine precursor loading. These parasympathetic ganglion cells did not show catecholamine fluorescence under control conditions. In the rat and mouse, but not in the guinea pig, some tracheal neurones were dopamine beta-hydroxylase immunoreactive and showed uptake and metabolism of amine precursors, thus expressing aspects of the catecholaminergic phenotype.

Some intrinsic neurons of the guinea-pig heart contain substance P.

Whole-mount preparations of the posterior wall of the atria of the guinea pig heart containing intrinsic ganglion cells and nerve plexuses were stained for substance P-like immunoreactivity by the peroxidase-antiperoxidase method. Substance P-like nerve fibres are present as pericellular baskets around most, but not all, of the neuronal cell bodies, and are also found in the connecting nerve bundles, as perivascular nerve plexuses and in the myocardium and pericardium. The majority of ganglion cell bodies are negative for substance P, as reported previously, but we describe for the first time, a small subpopulation of intrinsic neuronal cell bodies which show immunoreactivity for substance P. Therefore, not all cardiac substance P nerves are extrinsic afferent fibres. At present, the physiological role of intrinsic substance P neurones is not clear.

Afferent nerve endings in the tracheal muscle of guinea-pigs and rats.

The trachea of guinea-pigs was stained as a whole-mount preparation with the zinc iodide-osmium technique. A distinct class of nerve endings was observed associated with the tracheal muscle. The endings, issued from myelinated fibres of the vagus nerve via the recurrent laryngeal nerve, are distributed on either side of the midline and ventral to the tips of cartilages. They are interpreted as afferent nerve endings that may correspond to slow adapting stretch receptors identified by physiological studies. Each nerve contributes predominantly, but not exclusively, to the receptors of the ipsilateral side. There are 120-180 receptors along the full length of the guinea-pig trachea, their density being higher at the cranial end. The receptors are variable in size and structural complexity, and, to some extent, also in spatial orientation, but distinct subtypes are not recognizable. Receptors of similar morphology and distribution are found also in the rat trachea. The receptors can also be visualized with a cytochrome oxidase method for nerve endings, but they do not stain with immunohistochemistry for the neuropeptides substance P, calcitonin gene-related peptide, vasointestinal polypeptide and neurotensin.

Dorsal root ganglion neuron development in chick and rat.

The aim of this study was to compare the morphological development of dorsal root ganglion neurons in embryonic and early postnatal chicks and rats. The three-dimensional architecture of neurons was observed in ganglia in situ and in dissociated neurons by scanning electron microscopy after removal of the capsule and connective tissue. The percentages of neurons at different developmental stages were determined. The general morphological changes in the chick resembled those in the rat but the timing was different. In both chick and rat, the majority of neurons were bipolar at early stages of development (embryonic day 6 in chick and day 14 in rats) and later underwent pseudo-unipolarization to become mostly unipolar neurons at hatching or birth. This maturation event started at an earlier stage in chick embryos than in rats, with 57% unipolar neurons in chick and only 7% in rat on embryonic day 14. However, just after hatching or birth, at day 22 of development, a larger proportion of immature unipolar neurons remained in chicks (13%) than in rats (3%). We conclude that these differences should be taken into consideration in designing experiments on dorsal root ganglion neurons grown in tissue culture.

Neurogenic inflammation. A model for studying efferent actions of sensory nerves.

Several lines of evidence suggest that sensory nerves of the carotid body have an efferent function in addition to their afferent function of conducting chemoreceptive impulses to the brain. However, it has been difficult to document the release of substances from sensory nerve terminals on glomus cells and to determine whether such an efferent function plays a role in chemoreception. By comparison, the phenomenon of neurogenic inflammation has been relatively easy to study in rats and guinea pigs and has proven to be an informative model system for analyzing efferent actions of sensory nerves. The main characteristic of neurogenic inflammation is plasma leakage. Chemical irritants that activate unmyelinated sensory nerves cause plasma leakage in the skin, respiratory tract, and other organs by triggering the release of substances from sensory nerve fibers. Substance P, which is synthesized and released by some sensory neurons, appears to be the main active mediator, although other tachykinins, calcitonin gene-related peptide, and perhaps other peptides may also participate. Neurogenic inflammation results from the action of substance P on NK1 receptors, as demonstrated by selective NK1 receptor agonists and antagonists. The NK1 receptors involved in plasma leakage are located on the endothelial cells of postcapillary venules and collecting venules. Within seconds of the activation of NK1 receptors by substance P, gaps form in the endothelium of target vessels. The endothelial gaps are transient, and the leak normally ends in a few minutes. However, the magnitude of the response can increase in pathological conditions such as Mycoplasma pulmonis infection in rats, which results in a chronic inflammatory disease of the respiratory tract. The infected airway mucosa becomes abnormally vascular as a result of angiogenesis, and the endothelial cells of the newly formed vessels express increased numbers of NK1 receptors and thus are abnormally sensitive to substance P. Studies of neurogenic inflammation not only have helped to understand the efferent actions of sensory nerves but also have given insight into the mechanism and consequences of inflammatory changes in endothelial cells and in the plasma leakage that follows.

Tissue-selective mast cell reconstitution and differential lung gene expression in mast cell-deficient Kit(W-sh)/Kit(W-sh) sash mice.

BACKGROUND: Mast cell-deficient Kit(W)/Kit(W-v) mice are an important resource for studying mast cell functions in vivo. However, because they are compound heterozygotes in a mixed genetic background and are infertile, they cannot be crossed easily with other mice. OBJECTIVE: To overcome this limitation, we explored the use of Kit(W-sh)/Kit(W-sh) mice for studying mast cell biology in vivo. RESULTS: These mice are in a C57BL/6 background, are fertile and can be bred directly with other genetically modified mice. Ten-week-old Kit(W-sh)/Kit(W-sh) are profoundly mast cell-deficient. No mast cells are detected in any major organ, including the lung. Gene microarrays detect differential expression of just seven of 16,463 genes in lungs of Kit(W-sh)/Kit(W-sh) mice compared with wild-type mice, indicating that resting mast cells regulate expression of a small set of genes in the normal lung. Injecting 10(7) bone marrow-derived mast cells (BMMC) into tail veins of Kit(W-sh)/Kit(W-sh) mice reconstitutes mast cell populations in lung, stomach, liver, inguinal lymph nodes, and spleen, but not in the tongue, trachea or skin. Injection of BMMC into ear dermis or peritoneum reconstitutes mast cells locally in these tissues. When splenectomized Kit(W-sh)/Kit(W-sh) mice are intravenously injected with BMMC, mast cells circulate longer and are found more often in the liver and inguinal lymph nodes, indicating that the spleen acts as a reservoir for mast cells following injection and limits migration to some tissues. CONCLUSION: In summary, these findings show that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice possess unique attributes that favour their use for studying mast cell functions in vivo.

Scanning electron microscopic studies of bullfrog sympathetic neurons exposed by enzymatic removal of connective tissue elements and satellite cells.

The ninth and tenth abdominal sympathetic ganglia of bullfrogs were studied by light microscopy and transmission and scanning electron microscopy after the removal of the connective tissue elements overlying the neurons. Digestion of tissues with trypsin and subsequent acid hydrolysis exposed the unipolar neurons, which remained covered by their satellite cells. The preganglionic innervation was visible on the proximal segment and axon hillock region of the postganglionic neurite. Clusters of small cells seen at the periphery of ganglia probably corresponded to groups of cells with abundant catecholamine-containing granules (SIF cells). Digestion with collagenase and protease removed some or all of the satellite cells in addition to the connective tissue. The true neuronal surfaces had short finger-like processes, whereas the external surfaces of satellite cells were smooth. Preganglionic nerve varicosities were clearly visible on the proximal segment of the postganglionic neurite, on the axon hillock and on the cell body of neurons. A few axonal varicosities were fractured to reveal the synaptic vesicles within. The possible effects of the distribution and glial ensheathment of nerve varicosities on their function are discussed.