Three-Dimensional Ultrastructure of Flower-Spray Nerve Endings in the Rat Carotid Sinus.

The flower-spray nerve endings are afferent nerve terminals in the carotid sinus that arise from carotid sinus nerve of glossopharyngeal nerve. However, the three-dimensional ultrastructural characteristics of flower-spray nerve endings and spatial relationships between the terminal parts and other cellular elements have not been fully understood. To elucidate their detailed relationship, backscattered electron imaging of serial sections was performed with a scanning electron microscope to produce a three-dimensional reconstruction of the flower-spray endings. The terminal parts of flower-spray endings were distributed horizontally approximately 5 µm outside the external elastic membrane in the tunica adventitia of the internal carotid artery. The three-dimensional reconstruction showed that the terminal parts of flower-spray endings were flat with irregular contours and were partially covered by the thin cytoplasmic processes of Schwann cells. The complex consisting of the nerve terminals and associated Schwann cells was surrounded by a multilayered basement membrane. The terminal parts of the endings were also surrounded by fibroblasts with elastic fibers and collagen fibrils. Secretory vesicles without an electron-dense core were observed in the terminal parts of the endings. The accumulation of vesicles just below the axonal membrane was observed in terminal parts not covered by Schwann cell cytoplasmic processes on both the luminal and basal sides. Swollen mitochondria, concentric membranous structures, and glycogen granule-like electron-dense materials were often noted in some of the terminal parts of the endings and the parent axon. Collectively, the present results suggest that flower-spray endings are baroreceptors because their morphology was similar to other mechanoreceptors. Furthermore, flower-spray endings may be affected by glutamate secreted in an autocrine manner.

Morphological evidence of reinnervation of the baroreceptive regions in sinoaortic-denervated rats.

1. The arterial baroreflex (ABR) plays an important role in the maintenance of the stability of blood pressure. Sinoaortic denervation (SAD) destroys the integrity of the reflex arc and produces severe organ damage in rats. However, partial recovery of ABR function has been observed following chronic denervation. The aim of the present study was to determine whether there was morphological evidence of reinnervation of the aortic arch and carotid sinus following SAD. 2. A substantial body of physiological and morphological evidence suggests that substance P (SP) may be a neurotransmitter contained in first-order sensory baroreceptor afferents; therefore, the patterns of vascular SP and neurofilament (NF) immunoreactive (IR) innervation of the aortic arch and carotid sinus were investigated in the present study. 3. Ten-week-old male Sprague-Dawley rats underwent SAD or sham operation. Whole mounts of carotid bifurcation and aortic arch were prepared for immunohistochemical study at various time points (1, 9 and 16 weeks after operation). 4. The results of computerized image analysis show that the mean density of NF- and SP-IR nerves of SAD rats 9 and 16 weeks after operation increased gradually and significantly compared with that of rats 1 week after operation. 5. In conclusion, the results indicate that there is reinnervation of the aortic arch and carotid sinus by NF- and SP-IR fibres in SAD rats, which may be the morphological basis for the partial restoration of ABR function over time after SAD.

Catecholamine release from isolated sensory neurons of cat petrosal ganglia in tissue culture.

The petrosal ganglion (PG) is entirely constituted by the perikarya of primary sensory neurons, part of which innervates the carotid body via the carotid sinus nerve (CSN). Application of acetylcholine (ACh) or nicotine (Nic) as well as adenosine 5'-triphosphate (ATP) to the PG in vitro increases the frequency of CSN discharges, an effect that is modified by the concomitant application of dopamine (DA). Since a population of PG neurons expresses tyrosine hydroxylase, and DA is released from the cat carotid body in response to electrical stimulation of C-fibers in the CSN, it is possible that DA may be released from the perikarya of PG neurons. Therefore, we studied whether ACh or Nic, ATP and high KCl could induce DA release from PG neurons in culture. Petrosal ganglia were excised from pentobarbitone-anesthetized adult cats, dissociated and their neurons maintained in culture for 7-21 days. Catecholamine release was measured by amperometry via carbon-fiber microelectrodes. In response to KCl, Nic, ACh or ATP application, about 25% of neurons exhibited electrochemical signals compatible with DA release. This percentage increased to 41% after loading the neurons with exogenous DA. The present results suggest that DA release may be induced from the perikarya of a population of PG neurons.

Elastin and mechanoreceptor mechanisms with special reference to the mammalian carotid sinus.

Light microscopic studies reveal that the carotid baroreceptor region in mammals, located at the origin of the internal carotid artery, has a preponderantly elastic structure and a thick tunica adventitia. Electron microscopy discloses the presence of sensory nerve endings within the parts of the tunica adventitia adjoining the preponderantly elastic zone of the internal carotid artery. Bundles of collagen fibres in the tunica adventitia form convolutions or whorls around the nerve terminals and often terminate on the surface of the elastic fibres or into the basement membranes of the neuronal profiles. It is concluded that the large content of elastic tissue in the tunica media of the baroreceptor region renders the vessel wall highly distensible to intraluminal pressure changes, and thereby facilitates transmission of the stimulus intensity to sensory nerve terminals. However, a change in the geometrical configuration of the bundles of collagen under the influence of elastic fibres may provide a better insight into the mechanisms of distortion of the baroreceptors related to and/or in contact with collagen fibres. In support of this is the demonstration of contact sites between collagen and elastic fibres.

Viscoelastic influence on wall and baroreceptors of rabbit carotid sinus.

This study examined multifiber baroreceptor nerve activity (BNA) as a function of carotid sinus wall distension in 19 rabbits. Analysis estimated mechanical or viscoelastic properties of the sinus wall and their influence on BNA. In six sinuses, properties were altered by treatment with the enzyme protease to remove the endothelium and with nifedipine to passively relax smooth muscle. Properties were estimated from dynamic and steady state wall response to a 45 mm Hg step increase and decrease in intrasinus pressure (ISP) of 20 min. Control wall response had fast and slow (creep) portions with a viscosity increase from 1,370 N(s)/m to 17,864 N(s)/m during step-up in ISP. Wall elasticity averaged 77 N/m; which estimated the relationship of force and change in steady state response. Control BNA response also had fast and slow (resetting) portions. A BNA and wall response relationship (BNA/m) was defined as transduction-gain (T-G) with proportional and dynamic components. In the subgroup, wall creep and baroreceptor resetting were abolished by protease treatment, suggesting an endothelial mediator which influenced sinus smooth muscle. Histology data indicated enzyme damage was limited to tunica intima tissues, and nifedipine did not block Ca2+ channels on neural structures. By comparison of responses before and after treatments the proportional component of T-G was equated to an elastic influence (1/E), with E = 7.5 x 10(-6) m/BNA, while the dynamic component was equated to a viscous influence (1/V), with V = 1.53 x 10(-4) m(s)/BNA. A simple but fundamental relationship for baroreceptor-tissue linkages was estimated by BNA/m = 1/(Vs + E), a first-order transfer function.

Electron microscopic structure and innervation of the carotid baroreceptor region in the rock hyrax (Procavia capensis).

Semi-thin plastic sections reveal that the carotid baroreceptor region in the rock hyrax comprising the origin of the internal carotid artery has a preponderantly elastic structure and a thick tunica adventitia. In contrast, the common carotid artery has a musculoelastic structure, whereas the cranial segment of the internal carotid artery (immediately distal to the baroreceptor areas) shows the features of a muscular artery. Electron microscopy discloses the presence of sensory nerve endings within the parts of the tunica adventitia adjoining the preponderantly elastic zone of the internal carotid artery. These nerve endings are characterized by varicose regions containing a large quantity of mitochondria. Bundles of collagen fibers in the tunica adventitia form convolutions or whorls around the nerve terminals and often terminate on the surface of the elastic fibers or into the basement membranes of the neuronal profiles. The large content of elastic tissue in the tunica media of the baroreceptor region may render the vessel wall highly distensible to intraluminal pressure changes. This, in turn, would facilitate the transmission of the stimulus intensity to the sensory nerve terminals located in the tunica adventitia. It is suggested that the stretching of elastic fibers may form the main mechanical event leading to the distortion of the associated nerve terminals. However, a change in the geometrical configuration of the bundles of collagen under the influence of the elastic fibers may provide a better insight into the mechanisms of distortion of the baroreceptors related to and/or in contact with collagen fibers.

Ultrastructure of carotid baroreceptors in the goat.

The initial segment of the occipital artery of the goat appears modified under the electron microscope; endothelial cells are low cuboidal, the tunica media contains many elastic lamellae, and there are few smooth muscle cells. Free afferent nerve endings are seen in this modified arterial wall. They closely resemble the presumptive baroreceptor endings reported in other mammals in their mitochondrial content and abnormal organelles and are interpreted as solely adapting baroreceptors. Special topographical relations between the endings and elastic or collagen fibres do not occur, but microfilaments are present in the vicinity of these endings. Presumptive adrenergic efferent endings are not found in the region of the baroreceptors. We suggest that this zone of the occipited artery is homologous to the carotid sinus.

Neurotensin-like immunoreactivity in presumptive baroreceptor neurons innervating the guinea pig carotid sinus.

This study was carried out to determine the ultrastructure and origin of previously described neurotensin-like immunoreactive (NT-LI) nerve fibres in the wall of the carotid sinus of the guinea pig. In our degeneration experiments, these NT-LI axons were unaffected by surgical sympathectomy but disappeared in response to transection of the carotid sinus nerve, thus suggesting a sensory origin. This assumption could be supported by combined retrograde tracing and immunohistochemistry. Primary afferent neurons of the sensory glossopharyngeal (petrosal) ganglion projecting to the carotid sinus were identified by injecting the retrograde fluorescent tracer, Fluoro-Gold, into the sinus wall, and some of these neurons displayed NT-LI. Within the carotid sinus, the terminals of these NT-LI sensory neurons exhibited ultrastructural features characteristic of baroreceptor endings, i.e. axonal swellings filled with mitochondria and closely associated to elastin. However, many endings also fulfilling the ultrastructural criteria for baroreceptors were devoid of immunolabelling. Thus, we conclude that the NT-LI terminals constitute a subgroup rather than the entire population of baroreceptor endings within the guinea pig carotid sinus. With respect to the established pharmacological effects of NT in guinea pig, we propose an involvement of NT-LI fibres in the modulation of baroreception at the peripheral level.

Carotid baroreceptor control of liver and spleen volume in cats.

We tested the hypothesis that the blood volumes of the spleen and liver of cats are reflexly controlled by the carotid sinus (CS) baroreceptors. In pentobarbital-anesthetized cats the CS area was isolated and perfused so that intracarotid pressure (Pcs) could be controlled while maintaining a normal brain blood perfusion. The volume changes of the liver and spleen were estimated by measuring their thickness using ultrasonic techniques. Cardiac output, systemic arterial blood pressure (Psa), central venous pressure, central blood volume, total peripheral resistance, and heart rate were also measured. In vagotomized cats, increasing Pcs by 100 mmHg caused a significant reduction in Psa (-67.8%), cardiac output (-26.6%), total peripheral resistance (-49.5%), and heart rate (-15%) and significantly increased spleen volume (9.7%, corresponding to a 2.1 +/- 0.5 mm increase in thickness). The liver volume decreased, but only by 1.6% (0.6 +/- 0.2 mm decrease in thickness), a change opposite that observed in the spleen. The changes in cardiovascular variables and in spleen volume suggest that the animals had functioning reflexes. These results indicate that in pentobarbital-anesthetized cats the carotid baroreceptors affect the volume of the spleen but not the liver and suggest that, although the spleen has an active role in the control of arterial blood pressure in the cat, the liver does not.

New technique to completely isolate carotid sinus baroreceptor regions in rats.

We developed a method by which we can completely isolate the carotid sinus baroreceptor regions in the rat. The carotid sinus baroreceptor region is exposed and, with the use of extra-fine forceps, a human hair is placed around and tied at the root of the bifurcation. This procedure occludes the external carotid artery and blood flow to the carotid body. An injector is then attached to a catheter in the common carotid artery. We introduce a cylindrical rubber plug into either the palentine or internal carotid artery. A second plug is introduced to occlude the other artery. In six of the eight rats studied, these procedures completely isolated the carotid sinus region. In those cases where a small leak persisted at a carotid sinus pressure of 180 mmHg, we introduced a small particle of the animal's own previously clotted blood. Carotid sinus pressure was either randomly changed between 40 and 180 mmHg in 20-mmHg increments or in sequential 20-mmHg steps from 40 to 180 mmHg while measuring the animal's pulsatile and mean blood pressures. Arterial pressure-carotid sinus pressure relationship indicates that there is a highly sigmoidal relationship between the two pressures. The peak gain of the carotid sinus reflex system had a range from 1.5 to 4.0 and a mean value of 2.07 +/- 0.08. Our data indicate that the rat exhibits a significant carotid sinus baroreceptor reflex response. This technique combined with other techniques will allow for the study of neural control of cardiovascular function in the rat.

Structure of the carotid sinus of the dromedary camel (Camelus dromedarius).

The morphology of the terminal segment of the common carotid artery of the camel was studied. It was found that this artery terminated by giving off a patent internal carotid artery and continuing as the external carotid artery. A presumptive carotid sinus was present at the region of origin of the internal carotid. Similar to that of other mammals, the carotid sinus of the camel was characterized by a comparatively thin vascular wall rich in elastic fibres. With the electron microscope, free (non-encapsulated) afferent endings, efferent endings, encapsulated axons and bundles of unmyelinated and myelinated nerve fibres were found in the wall of the presumptive carotid sinus. The free afferent endings closely resembled baroreceptor endings of the carotid sinus of other mammalian species in their mitochondrial content and the presence of abnormal organelles such as lamellar bodies, vesicular mitochondria and dense bodies. They were regarded as slowly adapting baroreceptors. The presumptive efferent endings were considered adrenergic whereas the encapsulated axons were thought to be the proximal parts of encapsulated sensory nerve endings.

Ultrastructure of calcitonin gene-related peptide- and substance P-like immunoreactive nerve fibres in the carotid body and carotid sinus of the guinea pig.

Previous studies have demonstrated that substance P- (SP) and calcitonin gene-related peptide-like immunoreactivities (CGRP-LI) coexist in sensory nerve fibres in the guinea-pig carotid body and carotid sinus. In the present study the ultrastructure of these nerve fibres was investigated by means of single- and double-labelling immunocytochemistry. In both, carotid body and carotid sinus immunoreactive fibres were unmyelinated axons of small diameter (0.12-0.56 microns). At the subcellular level, SP- and CGRP-LI were colocalized in intra-axonal dense core vesicles, suggesting corelease and simultaneous action of these two compounds. SP/CGRP-LI nerve fibres within the carotid body were mainly found in the interparenchymal connective tissue, but also occurred in relationship to blood vessels and nests of glomus cells. Neither in the carotid body not in the carotid sinus, SP/CGRP-LI axons corresponded to the large terminals which are generally considered to represent the main chemoreceptor and baroreceptor endings, respectively. Thus, SP/CGRP-LI fibres either belong to the chemo- and baroreceptors of the C-fibre class or constitute a fibre population not directly involved in conduction of baro- and chemoreflexes.

Observations on the fine structure of the baroreceptors and adrenergic innervation of the guinea-pig carotid sinus.

We examined the fine structure of the baroreceptors and the adrenergic innervation of the guinea-pig carotid sinus. The tunica adventitia contained many nerve bundles whose perineuria enclosed unmyelinated nerve fibers, alone or together with myelinated nerve fibers. Baroreceptors, which lay close to elastic and collagen fibers in the adventitia and media, were surrounded by "terminal" cells with ultrastructural features characteristic of Schwann cells and contained inclusions of various types. Morphologic features of the baroreceptors included densely packed mitochondria, osmiophilic lamellated and homogeneous bodies, clear and granular vesicles, lamellar systems, glycogen granules, neurofilaments, neurotubuli, and vacuolated mitochondria. In animals that had been treated with 6-hydroxydopamine, occasional electron-dense endings (or fibers) were observed in the adventitial layer. The baroreceptors in the guinea-pig carotid sinus appear to have most of the morphologic features reported for other species.

Observations on the structure and innervation of the presumptive carotid sinus area in the giraffe (Giraffa camelopardalis).

The tunica media of the giraffe carotid artery is characterised by a predominantly muscular structure, except for a small area at the origins of the occipital and the vestigal internal carotid arteries. The latter has a preponderantly elastic structure and corresponds in extent to the parts of the carotid arterial wall innervated by a branch of the glossopharyngeal nerve. Sensory nerve terminals, characterised by an abundance of mitochondria, are found in the deeper parts of the adventitia bordering the elastic zone. It is concluded that the elastic zone at the cranial portion of the carotid arterial system in the giraffe constitutes the equivalent of a carotid sinus. It is further suggested that the preponderance of elastic tissue in this area is a morphological adaptation of the arterial wall to a baroreceptor function. Pertinent to this suggestion is the demonstration of a close structural relationship between the sensory nerve endings and the elastic fibres.

Neurotensin and substance P immunoreactive nerve endings in the guinea pig carotid sinus and their ultrastructural counterparts.

The carotid sinus of the guinea pig was analysed immunohistochemically for the occurrence of neuropeptides. Immunoreactivity (IR) for neurotensin (NT) and substance P (SP) is distributed in two different populations of nerve endings and varicosities. NT-IR fibers penetrate deeply into the tunica media of the elastic segment of the carotid sinus and form the large, branched lanceolate nerve terminals. Electron-microscopic investigations have revealed that the NT-IR varicosities correspond to the large afferent baroreceptor endings containing abundant mitochondria. SP-IR fibers are located mainly at the media-adventitial border. They seem to be correlated to dense-core, vesicle-containing varicosities identified in the electron microscope. Therefore, these fibers may constitute afferent and efferent perivascular plexus regulating the vascular tone of the carotid sinus wall.

Structure of the glomic arteries.

The structure of the arteries which supply blood tp the carotid bodies was examined in five cases in which the weights of the cardiac ventricles were normal and in which hypoxaemia due to emphysema or systemic hypertension were absent. The right glomic arteries were studied by light microscopy, and the left by electron microscopy. A sixth case, also free from myocardial hypertrophy, was used solely for an examination of the common carotid artery, the carotid sinus and the internal carotid artery by light and electron microscopy. The glomic arteries resembled the carotid sinus in being highly elastic with a rich supply of non-myelinated nerve fibres and thus had a structure consistent with having a baroreceptor function.

Electron microscopy of selectively denervated canine carotid sinuses.

Selective denervation as used to characterize the nerve endings in the carotid sinus wall of the dog. Areas of dense innervation were identified by acetylcholinesterase histochemical technique and sampled for electron microscopic study following survival periods of 48 to 144 hours after selective neurectomies were done. Large varicosities, similar to previously described mechanoreceptor endings, showed signs of degeneration after transection of the sinus nerve, but not after cranial cervical ganglionectomy or section of the glossopharyngeal nerve proximal to its sensory ganglia. These were concluded to be endings of afferent neurons. Histochemical reactions at the fine structure level demonstrated such varicosities to be acetylcholinesterase positive. Smaller varicosities containing granulated synaptic vesicles degenerated after cranial cervical ganglionectomy was done. These were interpreted to be postganglionic sympathetic endings. This study represents an example where denervation, coupled with short survival periods, serves as a technique for studying selected areas of the peripheral nervous system.

Ultrastructure of baroreceptors in the carotid sinus of the rabbit.

In the rabbit, baroreceptive intercalated and terminal receptor segments of the sinus nerve extend in the border region of adventitia and media of the carotid sinus wall. these receptors measuring 600-6,500 nm harbour a great number of mitochondria. They are either round, avoid or elongated. Round receptors are present over the whole area of the carotid sinus. Large receptors appear to be missing in the upper region, where only very elongated receptors are present, which are arranged in circular array in relation to the vascular axis. The baroreceptors are enveloped by special organelle-rich terminal cells of Schwann cell origin. The receptors have a special orientation in regard to the muscle-elastic system and to collagen. Receptors contain osmiophilic and laminated bodies, signs of degeneration and vesicles of different sizes. Many tubuli as well as glycogen granules are apparent. Some of these axoplasmatic changes appear to be related to metabolic processes (turnover) occurring in young and old animals.

Glomus tissue in the vicinity of the human carotid sinus.

Three of 60 cadavers have shown, in the adventitia or in the adipose tissue from the human carotid sinus region, small islands of tissue richly and typically vascularized and with nerve endings contacting cells like the tissue of the principal carotid body. In two of the cases such 'miniglomera' were single but in the third there were several all on the same side. A modified en bloc silver nitrate reduction stain was used to demonstrate the microvascular arrangements and the nerve endings by light microscopy of serial tangential sections of the carotid bifurcation.