Use of the peritracheal fold in the dog tracheal transplantation model.

OBJECTIVE: To investigate the technical aspects of the canine model of human tracheal transplantation for potential application to reconstruction of extremely long tracheal defects (> 10 cm). DESIGN: In phase 1, long tracheal segments were skeletonized and pedicled with the thyroid glands, cranial thyroid arteries and veins, and internal jugular vein branches. The segments were elevated completely, attached to the vascular pedicle only, and replaced with primary tracheal anastomoses. In phase 2, long segments were elevated along with a diffuse soft tissue "blanket" that envelops the trachea and thyroid glands. Because this study was designed to primarily address, in situ, tracheal perfusion territories of a cranially located vascular pedicle, microvascular anastomoses were not conducted. SUBJECTS: Two small-bodied beagles (10-15 kg) and 5 large-bodied mixed-breed dogs (20-30 kg) were humanely killed 2 to 41 days after surgery, and anatomic and histological analyses were conducted. RESULTS: Unlike that of humans, the thyroid gland complex of dogs is not intimately associated with the trachea but is conjoined with a peritracheal soft tissue "fold." Within this fold, blood is transmitted to the trachea via a diffuse, segmental vascular plexus. In phase 1, pronounced tracheal necrosis occurred within 2 to 5 days. In phase 2, extremely long tracheal segments (10-12 cm), based only on a cranially located pedicle, were still viable at 2 to 6 weeks. CONCLUSIONS: Preservation of the "peritracheal fold" in the dog model of tracheal transplantation is critical to the onset and maintenance of vascular perfusion in a long tracheal segment. Furthermore, the use of large-bodied dogs is necessary to provide for a usable venous efflux component.

The human aerodigestive tract and gastroesophageal reflux: an evolutionary perspective.

In order to appreciate fully the nature of supraesophageal complications of gastroesophageal reflux in humans, it is essential to view the problem within an evolutionary framework. Examination of the aerodigestive tract anatomy of our mammalian relatives shows that this region in humans is highly derived as compared to other mammals. Among the specializations that adult humans exhibit is a caudal position of the larynx, which results in a permanently expanded oropharynx. These anatomical features underlie our distinctive breathing and swallowing patterns and provide the substrate that allows for the production of articulate speech. While the selection factors that have shaped human evolution obviously favored our derived aerodigestive tract, aspects of this anatomy appear particularly unsuited to accommodate gastroesophageal reflux. Indeed, our unique aerodigestive tract morphology may predispose us to an array of supraesophageal complications of gastroesophageal reflux.

Use of the novel carbocyanine tracer fast-DiI for investigating upper respiratory tract cranial nerves in prenatal rats.

The use of traditional neuroanatomic tracing methods with tracers such as horseradish peroxidase requires living systems to take up and transport the label. These tracers have limited application in the study of prenatal systems. The advent of the carbocyanine dye DiI provided a tool by which neuronal pathways may be traced in postmortem fixed tissue. This dye allows careful dissection of prenatal organisms and specific application of the tracer to the neuroanatomic structure under investigation. Although DiI has become increasingly popular, it is limited by the difficulty in applying crystals to peripheral nerves and in the lengthy trace times, particularly in fixed tissue. A chemically modified version of DiI has been introduced that may overcome some of the limitations in using this tracer. The newer dye, fast-DiI, is easy to apply and can trace neural pathways in a shorter period. We describe our experience with the use of fast-DiI in the prenatal rat for the investigation of motoneurons that subserve upper respiratory tract structures. We have determined protocols for fixation, application of dye, processing of tissue, and visualization of traces. The entire protocol can be completed within 1 week, and the use of fast-DiI is easy to learn and apply. The resultant labeling of traced nerves is specific and clearly demonstrates respective motor nuclei and individual motoneurons.

Naturally occurring motoneuron cell death in rat upper respiratory tract motor nuclei: a histological, fast DiI and immunocytochemical study in the hypoglossal nucleus.

We have previously reported on our investigation of motoneuron cell death (MCD) in the rat nucleus ambiguus (NA). This article focuses on the other major upper respiratory tract motor nucleus: the hypoglossal. The hypoglossal nucleus (XII) contains motoneurons to the tongue and, as such, plays a critical role in defining patterns of respiration, deglutition, and vocalization. Motoneuron counts were made in XII in a developmental series of rats. In addition, the neural tracer fast DiI was used to ensure that all hypoglossal motoneurons had migrated into the nucleus at the time cell death was assessed. Furthermore, an antibody to gamma-aminobutyric acid (GABA) was used to determine the potential effect of inadvertently counting large interneurons on motoneuron counts. Cell death in XII was shown to occur entirely prenatally with a loss of 35% of cells between embryonic day 16 (E16) and birth. Fast DiI tracings of the prenatal hypoglossal nerve indicated that all motoneurons were present in a well-defined nucleus by E15. Immunocytochemical staining for GABA demonstrated considerably fewer interneurons than motoneurons in XII. These findings in XII, in comparison with those previously reported for NA, demonstrate differences in the timing and amount of cell death between upper respiratory tract motor nuclei. These differences establish periods during which one nucleus may be preferentially insulted by environmental or teratogenic factors. Preferential insults may underlie some of the upper respiratory tract incoordination pathologies seen in the newborn such as the sudden infant death syndrome (SIDS).

Naturally occurring motoneuron cell death in rat upper respiratory tract motor nuclei: a histological, fast DiI and immunocytochemical study of the nucleus ambiguus.

The mammalian upper respiratory tract (URT) serves as the common modality for aspects of respiration, deglutition, and vocalization. Although these actions are dependent on coordinated and specific neuromuscular control, little is known about the development of URT control centers. As such, this study investigated the occurrence of naturally occurring motoneuron cell death (MCD) in the nucleus ambiguus (NA) of a developmental series of rats. Standard histological techniques were used to count motoneurons in the ventrolateral brainstem where the mature NA is found. In addition, the neural tracer, fast DiI, was used to determine whether motoneurons were still migrating into the region of the NA during the period that cell counts were first taken. Furthermore, to elucidate the potential effect of inadvertently counting large interneurons on the assessment of motoneuron numbers, an antibody to gamma-aminobutyric acid (GABA) was used. The results of this study have, for the first time, demonstrated that MCD occurs in a URT-related motor nucleus. Approximately a 50% cell death was observed during the prenatal development of NA, with no further loss seen postnatally. The fast DiI studies showed that by embryonic day 17, NA was fully formed, suggesting that motoneuron migration from the basal plate was complete. In addition, use of the GABA antibody showed a lack of inhibitory interneurons within the NA. The finding of MCD in the NA helps define a critical period in the formation of URT neuromuscular control. As the course of MCD is modifiable by epigenetic signals, insult to the organism during this prenatal period may compromise future URT control.

Functional compartments of the tensor veli palatini muscle.

OBJECTIVE: To evaluate a dual role for the tensor veli palatini muscle in tubal and palatal function. MATERIALS AND METHODS: The eustachian tube region of guinea pigs and macaques was fixed and processed for analysis by serial section histologic examination, micro-dissection, or both. The attachment, fiber direction, and regional relations of potentially discrete functional compartments in eustachian tube muscles were noted. RESULTS: The tensor veli palatini muscle in macaques has two anatomic sub-bellies. One appears to be a tubal dilator, the other to make the tube rigid along its longitudinal axis. No other muscle is directly associated with the eustachian tube in macaques. The tensor veli palatini muscle in guinea pigs has one gross belly that may affect palatal tensing and tubal dilation, rigidification, and stabilization. Other muscles of the eustachian tube in guinea pigs are a medial sub-belly of the medial pterygoid muscle, not previously described, and the levator veli palatini muscle. CONCLUSIONS: The muscular elements underlying tubal-palatal function in guinea pigs are more distinct and spatially separated than in macaques or humans. These differences may explain the confusion about the role of accessory muscles in tubal function. Muscular compartments of the eustachian tube complex in guinea pigs are easily accessible, which facilitates a more discrete and confident placement of electromyography electrodes. The guinea pig may be a useful model to better understand the interaction of multilevel compartmentalized physiologic sequences that underlie coordination of swallowing, breathing, and middle ear aeration.

Anatomy of the hyoid apparatus in Odontoceti (toothed whales): specializations of their skeleton and musculature compared with those of terrestrial mammals.

BACKGROUND: The hyoid apparatus of odontocetes (toothed whales) serves as a major attachment point for many of the muscles and ligaments subserving breathing, swallowing, and sound production. METHODS: This study examines the hyoid apparatus in 48 specimens of ten odontocete genera (Phocoena, Lagenorhynchus, Stenella, Delphinus, Tursiops, Grampus, Globicephala, Mesoplodon, Physeter, and Kogia) collected post mortem from beach strandings. RESULTS: The odontocete hyoid apparatus, as that of their closest terrestrial relatives, the artiodactyls, is divisible into a basal portion (basihyal, paired thyrohyals) and a suspensory portion (paired ceratohyals, epihyals, stylohyals, and tympanohyals) connecting the basal portion to the skull base. Unlike other terrestrial mammals, the basal portion lies inferior to the laryngeal aditus, is flattened dorso-ventrally, and is relatively large, thus providing a broad surface area for muscle attachments. The suspensory elements are not as flattened and are joined by synovial joints (except for epihyal-stylohyal fusion). Muscular specializations include enlargement of those which retract the hyoid apparatus (e.g., sternohyoid) or control the tongue (e.g., styloglossus, hyoglossus). These muscles may be particularly important in a specialized prey capture behavior called suction feeding. In addition, the hyoid apparatus has a tilted placement, which allows asymmetrical enlargement of the piriform sinuses. Asymmetry is also seen in the muscular attachments between the larynx and the hyoid apparatus. The most pronounced differences from the basic pattern are observed in two families: Physeteridae and Ziphiidae. CONCLUSIONS: The derived position and shape of the odontocete hyoid apparatus may have evolved to subserve several specialized upper respiratory/digestive tract functions, such as simultaneous feeding (suction and swallowing) and sound production.

Specializations of the human upper respiratory and upper digestive systems as seen through comparative and developmental anatomy.

The human upper respiratory, or aerodigestive, tract serves as the crossroads of our breathing, swallowing and vocalizing pathways. Accordingly, developmental or evolutionary change in any of these functions will, of necessity, affect the others. Our studies have shown that the position in the neck of the mammalian larynx is a major factor in determining function in this region. Most mammals, such as our closest relatives the nonhuman primates, exhibit a larynx positioned high in the neck. This permits an intranarial larynx to be present and creates largely separate respiratory and digestive routes. While infant humans retain this basic mammalian pattern, developmental descent of the larynx considerably alters this configuration. Adult humans have, accordingly, lost separation of the respiratory and digestive routes, but have gained an increased supralaryngeal region of the pharynx which allows for the production of the varied sounds of human speech. How this region has changed during human evolution has been difficult to assess due to the absence of preserved soft-tissue structures. Our studies have shown that the relationship between basicranial shape and laryngeal position in living mammals can be a valuable guide to reconstruct the region in ancestral humans. Based on these findings we have examined the basicrania of fossil ancestors--from over two million years ago to near recent times--and have reconstructed the position of the larynx and pharyngeal region in these early forms. This has allowed us insight into how our ancestors may have breathed and swallowed, and when the anatomy necessary for human speech evolved.

Effect of basicranial flexion on larynx and hyoid position in rats: an experimental study of skull and soft tissue interactions.

The mammalian upper respiratory tract is a functionally dynamic region involved in respiration, deglutition, and phonation. As the structures of this area (e.g., larynx, hyoid) are suspended from the basicranium, changes in basicranial shape may affect both their anatomy and function. Although skeletal/soft tissue relationships have been examined through descriptive, comparative anatomy, these relationships have remained largely unexplored via experimental study. In this study, mechanical relationships between basicranial shape and positions of the larynx and hyoid bone are investigated experimentally. Skull base flexion was induced by surgically ablating the spheno-occipital synchondrosis in 13-day-old rats. Lateral radiograms were taken at 40, 60, 80, 100, and 120 days, and angular measurements made of basicranial shape and positions of the larynx and hyoid bone. Statistical analysis shows significant differences between experimental and control groups for skull base, hyoid, and larynx angles, and negative (inverse) correlations for basicranial shape change vs. hyoid position, and basicranial shape change vs. larynx position. Results show induced basicranial flexion caused inferior displacement of the larynx and hyoid bone, thus indicating a direct, mechanical relationship between skeletal and soft tissues. These observations may aid in understanding the basic biological, pathological, and evolutionary interactions of hard and soft tissues of the upper respiratory region.

Ultrasound investigation of fetal human upper respiratory anatomy.

Although the human upper respiratory-upper digestive tract is an area of vital importance, relatively little is known about either the structural or functional changes that occur in the region during the fetal period. While investigations in our laboratory have begun to chart these changes through the use of postmortem materials, in vivo studies have been rarely attempted. This study combines ultrasonography with new applications of video editing to examine aspects of prenatal upper respiratory development. Structures of the fetal upper respiratory-digestive tract and their movements were studied through the use of ultrasonography and detailed frame-by-frame analysis. Twenty-five living fetuses, aged 18-36 weeks gestation, were studied in utero during routine diagnostic ultrasound examination. These real-time linear array sonograms were videotaped during each study. Videotapes were next analyzed for anatomical structures and movement patterns, played back through the ultrasound machine in normal speed, and then examined with a frame-by-frame video editor (FFVE) to identify structures and movements. Still images were photographed directly from the video monitor using a 35 mm camera. Results show that upper respiratory and digestive structures, as well as their movements, could be seen clearly during normal speed and repeat frame-by-frame analysis. Major structures that could be identified in the majority of subjects included trachea in 20 of 25 fetuses (80%); larynx, 76%; pharynx, 76%. Smaller structures were more variable, but were nevertheless observed on both sagittal and coronal section: piriform sinuses, 76%; thyroid cartilage, 36%; cricoid cartilage, 32%; and epiglottis, 16%. Movements of structures could also be seen and were those typically observed in connection with swallowing: fluttering tongue movements, changes in pharyngeal shape, and passage of a bolus via the piriform sinuses to esophagus. Fetal swallows had minimal laryngeal motion. This study represents the first time that the appearance of upper airway and digestive tract structures have been quantified in conjunction with their movements in the living fetus.

A new surgical approach to the skull base in rats.

A new procedure is described to surgically approach the rat skull base and visualize the spheno-occipital synchondrosis. This region is difficult to assess in small laboratory animals without incurring damage to midline structures. This method, however, uses a lateral approach through an anterior triangle of the neck bounded by the sternocleidomastoid, digastric (posterior belly) and omohyoid muscles. No structures are transected in this procedure, thus preserving the underlying anatomy. This oblique angle of approach allows clear visualization of the mid-basicranium while sparing retraction or surgical trauma to respiratory and digestive structures such as the larynx, trachea and esophagus.

Mechanisms of middle ear aeration: anatomic and physiologic evidence in primates.

Proper aeration is a prerequisite for normal middle ear function in all terrestrial mammals. Our previous studies in primates provided anatomic evidence of neural circuits between the middle ear, brain, and eustachian tube by which central respiratory neurons can control middle ear aeration. Yet mechanisms that regulate middle ear aeration remain poorly understood. This study extends our research by examining maturation of these neural circuits, and investigating their underlying physiology. Ultrastructural examination of tympanic nerves, the afferent limb of the neural circuit, in an age-graded series of cynomolgus monkeys (Macaca fascicularis) showed substantial differences between newborn, young, and adult animals. These included a twofold increase in average myelin thickness, and greater than threefold increase in the ratio of myelinated to unmyelinated fibers from newborn to adult animals. These marked developmental changes may translate into functional differences in regulation of middle ear aeration in young animals, and possibly explain the extraordinarily high incidence of middle ear disease in early childhood. In physiologic experiments, bilateral electromyographic responses were recorded from eustachian tube muscles, the efferent limb of the neural circuit, in adult monkeys after ipsilateral stimulation of the tympanic nerve. Response latencies were 9 to 28 msec, similar to those of other multisynaptic bilateral brainstem reflexes. These physiologic data strongly suggest a concept of active control of middle ear aeration by respiratory neurons in the brain.

A new method for radiographically locating upper respiratory and upper digestive tract structures in rats.

A new procedure is described for radiographically locating structures of the upper respiratory and upper digestive tracts. These structures are difficult to visualize in small laboratory animals. In this method, a barium paste and tantalum dust mixture is specifically applied to the epiglottis, valleculae, soft palate and tongue in anesthetized rats to identify their positions. The mixture is easily visualized in radiograms and offers many advantages over traditional techniques, such as liquid barium swallows, insufflated tantalum powder or solid metallic implants. This enhanced visibility is due to the mixture's viscosity, which allows it to remain on the structures longer despite copious salivary flow. In addition, the thick consistency allows the radiopaque marker to adhere only where placed initially, and thus enables more precise measurements of the shape and position of upper respiratory and upper digestive tract structures.

The subarcuate fossa and cerebellum of extant primates: comparative study of a skull-brain interface.

The subarcuate fossa of the petrosal bone houses the petrosal lobule of the cerebellar paraflocculus. Although the subarcuate fossa can be extensive, little is known about its relative size and distribution in primates. Studies indicate parafloccular involvement with cerebellar areas coordinating vestibular, visual, auditory, and locomotor systems. Hypotheses have proposed a role for the paraflocculus in vestibular-oculomotor integration, caudal muscle control, autonomic function, and visual-manual predation. This study examines the morphology and relative extent of the subarcuate fossa/petrosal lobule in a range of living primates. Methods include study of postmortem specimens representing nine mammalian orders, and qualification of the volume of the subarcuate fossa and endocranial cavity in 155 dry primate crania of 36 genera. Results show that, in mammals, the size and morphology of the petrosal lobule is directly related to that of the subarcuate fossa. Craniometric analysis shows that the ratio of subarcuate fossa volume to endocranial volume is largest in lemuriforms. The largest ratio is in Microcebus and Hapalemur. Lorisids show a significant reduction in the size of the subarcuate fossa to almost 50% below the lemuriform mean. Tarsius is near the lemuriform mean. Among platyrrhines, the ratio is high, but significantly reduced compared to lemuiforms. The highest platyrrhine ratio is seen in Ateles, the lowest in Saimiri and Alouatta. Atelids are significantly elevated compared to cebids. In cercopithecids, the fossa is significantly reduced compared to platyrrhines. The trend toward reduction of the cercopithecid fossa is most pronounced in Theropithecus and least evident in Presbytis. In hominoids, the fossa is present only in Hylobates. In great apes and humans, other than Gorilla, the petromastoid canal occupies a similar location to the subarcuate fossa of other primates, but is not homologous to it. Neither the subarcuate fossa nor the petromastoid canal are present in Gorilla. A graded reduction of the subarcuate fossa/petrosal lobule is evident among primates which evolved later in time. The relative size of this cerebellar lobule within primates may reflect size-related factors and/or degree of neocortical evolution as these relate to usage of a specific sensory-mediated locomotor behavior. The subarcuate fossa may serve as an indicator to the differentiation of the petrosal lobule of the paraflocculus in fossil forms.

Existence of vocal folds in the larynx of odontoceti (toothed whales).

Odontocetes (toothed whales) vocalize for communication and echolocation. The mechanisms of sound production, however, remain unclear. Their larynx has long been thought to lack vocal folds and, thus, was considered incapable of generating sounds. This study investigates internal anatomy of the odontocete larynx to: 1) describe the morphology of any folds found, 2) determine any structural homologies between these folds and the vocal folds of terrestrial mammals, and 3) assess their possible function in sound production. Larynges of 24 odontocetes representing ten genera (Delphinus, Stenella, Lagenorhynchus, Tursiops, Grampus, Delphinapterus, Globicephala, Kogia, Mesoplodon, and Phocoena) were studied post mortem. Nine specimens were cut midsagittally, and the remainder were dorsally opened to reveal internal anatomy. Results show that, contrary to established belief, vocal folds are consistently present. They are not isolated bands or "cords," but appear continuous with the internal laryngeal membrane. The attachments of these folds are the same as in terrestrial mammals, thus indicating homology with true mammalian vocal folds. These folds extend from the midline of the thyroid cartilage to the base of the arytenoid cartilages, sometimes to a discrete process. The vocal folds are elongated and oriented in a vertical plane, parallel to airflow direction. Vocal fold morphology varies, appearing as true bifurcated structures, a trifurcated fold, or a single midline fold. Laryngeal ventricles and vestibular folds are also consistently found lateral to the vocal folds. The vocal folds may divide the airstream within the larynx into three separate air currents. Fold vibrations may produce initial laryngeal sound used in echolocation or communication.

Position of the larynx in odontoceti (toothed whales).

This study examines the positional relationships of the odontocete (toothed whale) larynx to further an understanding of their breathing, swallowing, and vocalizing abilities. Seventeen specimens representing nine cetacean genera (Delphinus, Stenella, Tursiops, Grampus, Delphinapterus, Globicephala, Kogia, Mesoplodon, and Phocoena) were studied post mortem. Nine specimens were sectioned in the midsagittal plane and the position of the larynx relative to vertebral levels, skull base, and palatal structures was recorded. In eight specimens that could not be bisected for reasons of large size or condition of preservation, the larynx was removed by a ventral approach for further dissection. The results show that the upper respiratory tract of the odontocetes has evolved away from a basic mammalian pattern. Laryngeal position among terrestrial mammals usually corresponds to the level of cervical vertebrae 1-3. The odontocete larynx, however, lies rostral to the level of the atlas and extends to the presphenoidal synchondrosis. Its extension above the level of the foramen magnum is due to three factors: 1) The larynx is elongated into a tubular extension that projects beyond the soft palate into the nasopharynx; 2) the neck region is shortened owing to the highly compressed cervical vertebrae; and 3) the skull base is oriented in the same direction as the cervical vertebrae because of the horizontal and fusiform alignment of the head and thorax. Whereas the larynx of most terrestrial mammals is separable from the nasopharynx, that of the odontocetes studied may be permanently intranarial, held in place by the palatopharyngeal sphincter. Laryngeal position may affect their vocal abilities, allowing odontocetes to simultaneously swallow and echolocate.

Developmental change in the position of the fetal human larynx.

The position of the mammalian larynx has been shown to be an important determinant in breathing, swallowing, and vocalizing patterns. While the growth of the adult human larynx has been studied extensively, detailed examination of fetal development has not been undertaken. Thus, crucial developmental change in the fetal period and the effects of this change on normal maturation are still unclear. This study has examined the development of the larynx and its topography during the fetal period. Thirty specimens were preserved in 10% buffered formaldehyde solution for a period of 6 weeks, after which mid-sagittal sections were performed. Fetal ages were calculated from femur diaphyseal lengths and ranged from 15 to 29 weeks. Direct measurements were taken to determine the growth and position of the larynx and trachea relative to the vertebral column and soft palate. Results show that the upper and lower levels of the larynx correspond to the basiocciput and the lower border of the third to upper border of the fourth cervical vertebrae, respectively. The epiglottic cartilage was present at 15 weeks. By 21 weeks, the epiglottis was well developed and in close palatal apposition. At 23 to 25 weeks, the epiglottis and soft palate were found to be in full contact. The acquisition of this contact may be related to fetal respiratory viability.