The structure, innervation and location of arteriovenous anastomoses in the equine foot.

In the foot of the horse, arteriovenous anastomoses (AVAs) of epithelioid type occurred in the dermis of the coronary band, in the coronary and terminal papillae, in neurovascular bundles and at the entrance to and along the length of the dermal laminae. A particular feature of the epithelioid segment of AVAs in the horse, compared with that of other species, was the height and surface complexity of many of the endothelial cells. They extended into the lumen, forming undercut and tunnel-like areas which correlated with the characteristic surface marking of AVAs observed in vascular casts. The number of cell organelles, including the concentration of vesicles in the luminal cytoplasm, suggested cells with a high metabolic activity. The luminal surface possessed numerous microvilli and long cytoplasmic cell processes which appeared to surround material in the lumen. The innervation of AVAs was more dense than that of the arteries and consisted of adrenergic and peptidergic nerves. Noradrenaline- and neuropeptide Y-containing nerves were identified as the vasoconstrictor components of the nerve supply and occurred along arteries and formed dense plexuses around AVAs. Calcitonin gene-related peptide, substance P and vasoactive intestinal polypeptide are vasodilators and were present in single nerve fibres which accompanied arteries and AVAs along the length of the dermal laminae. In this study the distribution, density and innervation of AVAs in the equine foot are correlated with their proposed role in the development of acute laminitis. The release of vasoactive peptides from diseased organs remote from the foot may induce inappropriate prolonged dilatation of AVAs and thus contribute to the laminar ischaemia of acute laminitis.

A scanning and transmission electron microscopic study of the development of the surface structure of neuroepithelial bodies in the mouse lung.

Neuroepithelial bodies (NEBs) are groups of neuroepithelial (NE) cells that are localized on mounds on the bronchiolar epithelium of the lung. The present study examined NEBs in mice ranging in age from 2 days before birth to 80 days after birth. The position and surface architecture of NEBs was examined using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In foetal mice, 2 days before birth, NEBs were distinguished from the rest of the bronchiolar epithelium by a slight elevation of non-ciliated Clara-like cells arranged in a cobblestone-like pattern. The exposed surface of the NEB was identified by small protrusions with regular microvilli intermittently located at the base of deep clefts between the Clara-like cells. The surface of the Clara-like cells had fewer and smaller microvilli and could be easily distinguished from the apical surface of the NEB. Before birth, the surface of all of the apical cells was covered by regularly placed microvilli, however after birth some of the more prominently positional apical cells revealed a bare patch at the centre of the portion of apical cell exposed to the lumen of the lung. As the mice aged there was an increase in the number of apical cell protrusions observed with centrally positioned bare patches. These two morphologically distinct surfaces of apical cells may have separate specialized functions. The exposed surfaces of apical cells were often observed in pairs and this feature has been observed in various sensory organs providing support for chemoreceptive function. However small bright spheres resembling vesicles were frequently observed on the lumenal surface of apical cells of the centrally placed bare patch. Transmission electron microscopy confirmed the presence of vesicles on the surface of apical cells and due to their location these vesicles were thought to contain a substance secreted into the lumen of the lung by apical cells. The significance of the bare region on the apical cells is not clear in terms of the proposed chemoreceptive function usually attributed to NEBs. It may be possible that the morphological changes observed in apical cells after birth are more appropriate for secretion of a substance into the lumen of the lung than for chemoreception. This is supported by the observation in the present study of vesicles lying on the lumenal surface of the bare region of the apical cell, however the mechanism for secretion of whole vesicles is not clear and requires further investigation.

Evidence for the coexistence of serotonin and calcitonin gene-related peptide at the subcellular level in neuroepithelial bodies in the lung of a marsupial, Isoodon macrourus.

The coexistence of serotonin and calcitonin gene-related peptide (CGRP) in neuroepithelial bodies of the bandicoot, Isoodon macrourus, has been examined using immunocytochemistry at the light- and electron-microscope levels. The avidin-biotin technique of antigen localisation was used initially to identify serotonin-like and CGRP-like immunoreactivity (-LI). Serotonin-LI and CGRP-LI were found in neuroepithelial cells in the lungs of 30-day-old bandicoots. CGRP-LI could also be demonstrated in nerve fibres associated with some neuroepithelial bodies. The protein A-gold technique of antigen localisation was used to label neuroepithelial cells and nerve fibres at the subcellular level. Serotonin-LI and CGRP-LI were observed in the same dense-cored vesicles of most neuroepithelial cells; however, some neuroepithelial cells were shown to possess serotonin-LI without CGRP-LI. Nerve fibres immediately adjacent to neuroepithelial bodies exhibited mainly CGRP-LI. These results show that serotonin-LI and CGRP-LI are present in neuroepithelial cells of the bandicoot in the same secretory vesicles. This pattern of co-localisation may reflect co-ordinated or synergistic actions of these two neuroactive substances.

Identification of B-epitopes in the human papillomavirus 18 E7 open reading frame protein.

A panel of murine mAb raised against a MS2 replicase/HPV 18 E7 fusion protein included 23 reactive by ELISA with HPV 18 E7 determinants. A total of 19 of the 23 recognized linear epitopes in the N-terminal region of the E7 molecule, while the other four were deduced by binding inhibition assays to recognize conformational determinants in this region. All tested antibodies precipitated a 14-kDa peptide doublet that corresponded with the predicted size of the E7 protein, from HeLa cells, but not from HPV 16 E7 containing CaSki cells. HPV 18 E7 protein was detected by immunolabeling with electron microscopy in both the nucleus and the cytoplasm of HeLa cells with the greater proportion occurring in the cytoplasm. No antibody reacted specifically by indirect immunofluorescence with HeLa cells. Weak cross-reactivity of some mAb with the E6 MS2-replicase fusion protein of HPV 16 was detected by ELISA, but no protein of the appropriate size was immunoprecipitated from CaSki cells. It is concluded that the B cell epitopes on the HPV 18 E7 transforming protein are located in the N-terminal region of the molecule and that some are weakly cross-reactive with HPV 16 E6 protein. E7 protein is either present in HeLa cells at a concentration too low to be detected by indirect immunofluorescence, or the N-terminal epitopes are masked by protein conformation or interaction with cellular or other viral components.

Peptide coexistence in axon terminals within the superficial dorsal horn of the rat spinal cord.

The somata of primary sensory neurons have been shown to contain up to four (and possibly more) neuroactive peptides. Although each of these peptides has been separately located in axon terminals within the superficial dorsal horn of the spinal cord, it is not clear whether multiple peptide coexistence is also a feature of terminal varicosities. The aim of this study was to determine whether the peptides substance P (SP) and calcitonin gene-related peptide (CGRP), which are colocalized in the somata of a large number of primary sensory neurons, coexist in the central terminals of these neurons in the spinal cord. The protein A-gold technique of antigen localization was used to screen single boutons in laminae I and II of the rats spinal cord for SP- and CGRP-like immunoreactivity at the ultrastructural level. The results show that SP and CGRP are colocalized within a large number of synaptic boutons in the superficial dorsal horn. Furthermore, evidence was obtained to suggest that both SP and CGRP may be found in the same synaptic vesicle within these boutons. These findings indicate that both SP and CGRP may be coreleased from single terminals in the superficial dorsal horn. This is of considerable interest in view of the reported interaction between SP and CGRP in nociceptive behavioral responses in the rat.

Innervation of arteriovenous anastomoses in the sheep tongue: immunocytochemical evidence for coexistence of neural transmitters.

In this study structural and immunocytochemical evidence has shown that arterial vessels, particularly AVAs, are associated with nerves containing peptidergic vasodilators, viz. VIP, CGRP and SP. The presence of VIP-like immunoreactivity in both P-type and C-type nerves is evidence of the coexistence of VIP and acetylcholine in cholinergic nerves and suggests the action of VIP in maintaining the opening of AVAs in heat stress conditions. The evidence for the co-existence of CGRP and SP is more direct as immunoreactivity for both peptides has been demonstrated in serial sections of the same nerve terminal. Although SP is a potent vasodilator there is little evidence of its role in thermoregulation; however it may be involved in a local axon reflex and cause antidromic vasodilatation of local vessels particularly AVAs.

Fate of senescent megakaryocytes in the bone marrow.

Degenerating senescent megakaryocytes have been identified in mouse bone marrow by light and electron microscopy. The ultrastructural changes which occur as degeneration proceeds are characteristic of death by apoptosis, although most cells appear to round up rather than undergo fragmentation. A hitherto unreported finding in degenerating cells was the presence of bundles of approximately 7 nm diameter parallel filaments in nuclei and membrane-bound nuclear fragments. Structurally, they resembled bundles of filaments induced in nuclei with dimethyl sulphoxide and identified as actin. Often a bundle appeared to terminate at the inner membrane. In the cytoplasm the presence of microtubules and centrioles indicates that not all the latter organelles are lost by the megakaryocyte during platelet release. Degenerating senescent megakaryocytes are rare in the marrow of normal mice but increase in frequency during 5-fluorouracil stimulated thrombocytosis. The dying cells are eventually phagocytosed by macrophages, a process that can occur extravascularly, showing that entry of senescent megakaryocytes into the circulation is not necessary for their disposal.

Time-lapse cinemicrography and scanning electron microscopy of platelet formation by megakaryocytes.

The surface architecture of megakaryocytes undergoing platelet formation in vitro has been examined by time-lapse cinemicrography and scanning electron microscopy. Fragments of mouse bone marrow were placed in culture medium and incubated at 37 degrees C. After several hours mature megakaryocytes migrated out of the marrow and some underwent shape changes so that they eventually appeared as a relatively small central body, housing the nucleus, from which emerged a number of thin processes which resembled platelet chains. Scanning electron microscopy showed that initially the megakaryocyte surface was ruffled but with development of processes it became smoother. Circumferential folds of small amplitude were found on the surface of developing constrictions which separated putative platelets. It is thought they may be associated with the mechanism of extension, but could have a role in establishing the topography of membrane components. Rupture of the chains and release of platelets was not observed; this permits the number of putative platelets formed by individual megakaryocytes to be determined. The putative platelets exhibited features common to circulating platelets when exposed to a glass surface including the development of pseudopodia and, eventually, flattening on to the surface.

The demarcation membrane system of the megakaryocyte: a misnomer?

The concept that the demarcation membrane system delineates platelets within the cytoplasm of megakaryocytes has been examined. In short-term culture of mouse bone marrow, mature megakaryocytes extended long, attenuated processes that were found by electron microscopy to have a limited amount of invaginated membrane. When such megakaryocytes were exposed to microtubule depolymerizing agents, the attenuated processes retracted, became thicker, and an extensive demarcation membrane reappeared. It is suggested from the results that the demarcation membrane system functions to provide a membrane reserve that undergoes evagination during the formation of attenuated processes and thereby envelops putative platelets, rather than to demarcate platelets in the maturing megakaryocyte. The term "invaginated membrane system" is considered more appropriate than "demarcation membrane system."

The ultrastructural characteristics of the apical cell in the neuroepithelial bodies of the toad lung (Bufo marinus).

The cytological features and membrane specialisations of neuroepithelial cells (apical cells) in direct contact with the lumen of the lung were studied with transmission and scanning electron microscopy. The luminal surface of the apical cell is characterised by microvilli, a cilium with an 8 + 1 microtubular pattern and numerous coated vesicles. The cytoplasmic region immediately beneath the luminal plasma membrane contains numerous smooth-walled vesicles, tubules and microtubules, a few microfilaments and dense granules (15-20 nm in diameter). The luminal pole of the cell is marked off from the basal or vascular pole by a well-defined terminal web associated with junctional complexes. Protrusion of the luminal pole occurs as a transient phenomenon and is accompanied by a pinching in of the cell at the terminal web. It is proposed that the distinctive features of the luminal pole of the apical cell are comparable to those of recognised chemoreceptor cells. It is also proposed that in view of the common features of apical and basal cells the apical cell functions as a receptor/transducer and the basal cells served as an accessory source of peptides/5-hydroxytryptamine to be released on stimulation of the apical cell. Furthermore, we have drawn attention to the structural heterogeneity of the neuroepithelial bodies in various vertebrate classes.

Ultrastructural identification of non-adrenergic, non-cholinergic nerves in the rat anococcygeus muscle.

The innervation of the rat anococcygeus muscle has been investigated ultrastructurally following fixation with a modified chromaffin reaction for the demonstration of biogenic amines (Tranzer and Richards, 1976). Three types of nerve profiles were revealed: (1) 60-70% of the profiles are adrenergic; (2) less than 5% of the profiles appear to be cholinergic; (3) up to 40% of the profiles are distinguished by the presence of a characteristically high proportion of electron-opaque, chromaffin-negative vesicles, 85-110 nm in diameter. This third type of profile was not affected by 6-OHDA, and is considered to represent the non-adrenergic, non-cholinergic inhibitory innervation of this tissue. Because of the morphological similarity of this nerve type, apart from the smaller vesicle size, to classical peptidergic nerve endings, they have been termed "small p-type" (sp-type). These results are discussed in relation to a previous report describing only two types of nerve profiles in this tissue (Gillespie and Lüllmann-Rauch, 1974).

Fine structural and cytochemical study of the innervation of smooth muscle in an amphibian (Bufo marinus) lung before and after denervation.

The innervation of the toad (Bufo marinus) lung was studied with transmission electron microscopy and fluorescence techniques, both before and after 12 or 20 days close vagosympathetic denervation. Four cytologically distinct types of neuronal processes were recognised, in relation to the visceral muscles of the lung. These were described as cholinergic, adrenergic, non-adrenergic/non-cholinergic (NANC) and sensory on the basis of the characteristics of their vesicular content and cytochemical reactions. An apparent efferent innervation of visceral smooth muscle was achieved by NANC (50%), cholinergic (25%) and adrenergic (25%) fibres. A few sensory fibres were also present. After denervation only NANC fibres persisted, showing that the cell bodies of these fibres were intrapulmonary. The vascular smooth muscle was supplied by cholinergic, adrenergic and sensory fibres. In the walls of the proximal branches of the pulmonary artery were fibres containing large dense-cored vesicles. These profiles, which were associated with the vasa vasorum, were similar to neurosecretory fibres. After denervation all neural profiles associated with the vasculature had degenerated. The observations suggest that vagal vasodepressor effects in the toad lung are mediated indirectly through relaxation of visceral muscle strands which in their contracted state compress vascular channels.

Innervation and cytochemistry of the neuroepithelial bodies in the ciliated epithelium of the toad lung (Bufo marinus).

Neuroepithelial bodies (NEB) were identified in the lung of Bufo marinus. The characteristics of the cells and their innervation were studied with electron and fluorescence microscopy before and after close vagosympathetic denervation. The bodies consist of low columnar cells which rest on the epithelial basal lamina. The majority of the cells do not reach the lumen of the lung (basal cells); the few which do (apical cells) are bordered by microvilli and possess a single cilium. The neuroepithelial cell cytoplasm contains a variety of organelles the most characteristic of which are dense cored vesicles. Microspectrofluorometry and electron microscopic cytochemistry indicate significant quantities of 5-hydroxytryptamine in these cells. The neuroepithelial bodies could be divided into three groups on the basis of their innervation: 1) About 60% of the NEBs are innervated solely by nerve fibers containing agranular vesicles which form reciprocal synapses; 2) about 20% are innervated solely by adrenergic nerve fibres which from distinct synaptic contacts; and 3) the remaining 20% are innervated by both types of nerve fibres. It is proposed that the NEBs are receptors monitoring intrapulmonary P CO2 and so leading to modulation of activity in afferent nerve fibres (type containing a granular vesicles). The presence of NEBs soley with an adrenergic (efferent) innervation poses a problem with this interpretation.

The innervation and fine structure of paraneuronic cells in an amphibian pulmonary artery.

The pulmonary artery of Bufo marinus contains large numbers of bipolar cells situated in the tunica adventitia and in the outer layers of the media. These cells show a bright green-yellow fluorescence (emission spectra 485 nm) after formaldehyde pre-treatment suggesting that they contain a primary monoamine. The most characteristic fine-structural feature of these cells is the presence of numerous dense-cored vesicles (80--300 nm diameter) in their cytopalsm. The cells are in close contact (20 nm gap) with both agranular and granular nerve fibres. Both EM-cytochemical and formaldehyde-induced fluorescence tests indicate that the granule-containing nerve fibres are adrenergic. The agranular nerve fibres form discrete synaptic contacts with pre and post-synaptic membrane thickenings on the cells. This was never observed with respect to the adrenergic fibres. Each process of the cells is about 45 micrometer long. The processes do not bear any special relationship to either vessels of the arterial vasa vasorum or medial smooth muscle cells. Their location in the wall of the artery suggests that they are functionally significant with respect to activity of the arterial media.