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.

Motor innervation of the eustachian tube muscles in the guinea pig.

The brainstem location of motoneurons innervating eustachian tube-associated muscles in the adult guinea pig was determined using intramuscular injections of the neural tracer horseradish peroxidase (HRP). Following HRP injections into the tensor veli palatini and the eustachian tube belly of the medial pterygoid muscle, an ipsilateral column of HRP-labeled motoneurons was present medial to the dorsolateral division of the trigeminal motor nucleus. Following HRP injection into the levator veli palatini, labeled motoneurons were present in the ipsilateral dorsal division of nucleus ambiguus. The locations of the tensor veli palatini and levator veli palatini motoneurons are similar to those found in studies of other animals. A distinct eustachian tube belly of the medial pterygoid muscle was also identified. This sub-belly had a motoneuron pool distinct from the main medial pterygoid muscle group. The authors have provided the gross anatomical and neuroanatomical substrates upon which future studies of eustachian tube function in the guinea pig may be based.

An external reference to identify the internal auditory canal in middle fossa surgery.

Traditional middle fossa landmarks, such as the facial hiatus and arcuate eminence, are often unreliable. This study was performed to establish an external reference from which to identify precisely the surgical anatomy of the middle fossa. The head of the malleus was identified from the middle fossa in the temporal bones of 20 adults. The lateral cortex of the temporal squamosa at the zygomatic root was used as the external plane of reference. The head of the malleus was consistently located 18 mm medial to the outer cortex on a line perpendicular to the reference plane. Medial extension of this line through the malleus head bisected the internal auditory canal. We recommend this method to precisely locate the malleus head as the first landmark in the middle fossa. Other structures, such as the geniculate ganglion, internal auditory canal, and superior semicircular canal can then be safely identified. We also present our results using this technique in six consecutive patients undergoing middle fossa surgery during the past 12 months.

Ethics and the care of persons with Bell's palsy.

Much of the bioethics literature focuses on dilemmas physicians face involving life and death issues. We articulate and apply a method of bioethics that can help both in resolving such dilemmas and in the appreciation of many situations and conditions. We apply the method to a case of a person with Bell's palsy because such a condition does not involve life and death, has a low cure rate, involves poorly recognized value conflicts, involves several specialties with rival approaches to care and much uncertainty. We show the strengths of the method in the application, and recommend it as generally useful to organize the way one perceives cases and attempts to resolve dilemmas.

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.

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.

Multiple head and neck neoplasia following radiation for benign disease during childhood.

A woman received radiation therapy to the adenoids for benign disease at the age of 10 years and subsequently developed an adenocarcinoma of the middle ear, a parathyroid adenoma, and a papillary carcinoma of the thyroid gland in adulthood. This appears to be the first such case on record. The literature of neoplasia after head and neck irradiation is briefly reviewed.

Neural control of middle ear aeration.

This study investigates the afferent and efferent pathways by which respiratory neurons in the brain can monitor and regulate middle ear aeration. Experiments were performed on 11 adult cynomolgus monkeys (Macaca fascicularis). The neural tracer, horseradish peroxidase, was placed on the transected nerves of the tympanic plexus in four animals. Horseradish peroxidase-labeled nerve terminal fields were observed in the ipsilateral respiratory subnuclei of the nucleus of the solitary tract. This may represent the sensory pathway by which the degree of middle ear aeration is monitored by the brain. Horseradish peroxidase was injected into the eustachian tube muscles in six of the monkeys, and horseradish peroxidase-labeled motoneurons were observed in the ipsilateral trigeminal motor nucleus (tensor palati muscle) and nucleus ambiguus (levator palati muscle). These brain-stem motor nuclei may represent the efferent pathways by which the degree of middle ear aeration is regulated. The results of these primate experiments confirm our earlier studies on rabbits and cats. A theory for the neural control of middle ear aeration is proposed.

A specialized innervation of the tensor tympani muscle in Macaca fascicularis.

The innervation of the tensor tympani muscle of the middle ear in Macaca fascicularis (cynomolgus monkey) was studied using the horseradish peroxidase (HRP) neural tracing technique. A compact column of small trigeminal motoneurons was labeled ipsilaterally following intramuscular application of HRP to the tensor tympani muscle. This column is located ventral and lateral to the dorsolateral division of the trigeminal motor nucleus, and just medial to the descending trigeminal nerve rootlets. No labeled neurons were present in the trigeminal mesencephalic nucleus or any other brainstem nucleus. Results are compared with those previously reported in several non-primate mammalian species, and in detail with that of the cat. A possible differential role of the tensor tympani muscle in acoustic modulation/middle ear aeration between primate and non-primate mammals is discussed.

Complications in the management of large glomus jugulare tumors.

This study analyzes the complications encountered in the surgical treatment of 17 patients with large glomus jugulare tumors. All 17 patients sustained either new cranial nerve palsies or exacerbation of preexisting palsies. These involved, in descending order of frequency, the facial nerve, the vagus and glossopharyngeal nerves, and the hypoglossal nerve. Postoperative palsies of the facial nerve involved 17 patients, as compared to 5 preoperatively. Fifteen patients had postoperative partial or complete paralysis of the vagus nerve as compared to eight preoperatively. Ten patients had postoperative palsies of the hypoglossal nerve as compared to six preoperatively. Other complications included CSF leak, meningitis, and wound infection. Aspiration and dysphagia were encountered postoperatively in 13 patients. Teflon injection of paretic vocal cords and cricopharyngeal myotomy effectively improved the ability to swallow and the quality of the voice. Prompt recognition and treatment of complications is essential for effective surgical management of large glomus jugulare tumors.

The monkey vertical vestibuloocular response: a frequency domain study.

We studied the vertical vestibuloocular response (VVOR) in seven cynomolgus monkeys. Eye movements were measured by the search coil method. We tested the monkeys by rotating them about their interaural axis, which was colinear with gravity. Each monkey was tested by using a standard rotational paradigm that consisted of discrete sinusoidal oscillations at three frequencies (0.01, 0.1, and 1.0 Hz) and six peak velocities (5, 10, 30, 60, 100, and 150 degrees/S). The standard rotational paradigm was applied twice for each of two conditions. The first condition (EOD) consisted of rotations with the animal's vision occluded; the second condition (EOL) consisted of rotations during which the animal was allowed to view a well-lighted room. Using various statistics, we tested the linearity of the sinusoidal slow-phase velocity component of the VVOR. The largest nonlinearity found was a skewness of approximately 14% in the waveform of f = 0.01 Hz. We did not find an amplitude asymmetry between slow-phase eye velocity upward (SPVU) and slow-phase eye velocity downward (SPVD) greater than 6% for any oscillation. Nonlinearities present in the VVOR during testing with vision occluded (EOD condition) disappeared with the addition of vision (EOL condition). Intensity function plots [peak slow-phase eye velocity vs. peak rotator (head) velocity] revealed that at f = 0.01, 0.1, and 1.0 Hz over the intensity range from +/-30 degrees/s to +/-150 degrees/s, the VVOR is highly linear. The lowest correlation coefficient associated with linear regressions of the intensity function data at each frequency was 0.99. Analyses of frequency response functions for the bandwidth f = 0.01 to 1.0 Hz, revealed the following: 1) mean amplitude ratio (AR) and phase overlap for four different stimulus intensities (30, 60, 100, and 150 degrees/s); 2) no significant differences (Mann-Whitney U test, P greater than 0.05) between any AR or phase value for mean peak SPVU and mean peak SPVD re appropriately directed head velocity; 3) no significant differences (Mann-Whitney U test, P greater than 0.05) between AR and phase values for animals tested and then retested 1 mo later with five intervening standard rotational paradigms; 4) a large effect of vision in producing a VVOR with near-unity gain and near-perfect phase compensation.(ABSTRACT TRUNCATED AT 400 WORDS)

A light and transmission electron microscope study of the neural processes within the pigeon anterior semicircular canal neuroepithelium.

This report addresses two questions. First, what is the incidence and distribution of multiple type I hair cells within a single NC in the pigeon's anterior semicircular canal crista? Second, are the synaptic structures found in the avian anterior crista similar to those found in the mammalian crista and if so are they different for single and multiple hair cell calyxes? Three pigeon anterior cristae were studied using interference LM and one pigeon anterior crista was studied using TEM. The light microscope studies showed that about 28% of the NCs studied contained a single type I hair cell; 64% contained 2-5 type I hair cells; and about 8% contained 6-12 type I hair cells. It was noted that the largest number of type I hair cells/unit area was located on the lower slopes of the crista while the smallest number of type I hair cells/unit area was located on the upper slopes and apex of the crista. The TEM studies showed that of 51 hair cells studied, 35% were type II hair cells and 65% were type I hair cells. These studies also showed that synaptic structures described in mammals are also seen in the pigeon. So far, in our preliminary studies we have been unable to demonstrate any difference in synaptic structures associated with single calyxes and those associated with multiple calyxes.

Primary adenomatous neoplasm of the middle ear.

Adenomatous lesions of the temporal bone are seldom encountered in clinical practice. Four cases of primary adenocarcinoma of the middle ear are reported. All four patients presented with a mass behind an intact tympanic membrane. Two of the four patients had received radiation to the head and neck area 10 or more years prior to their developing adenocarcinoma of the middle ear space. Although this tumor is locally invasive, it does not appear to be highly aggressive and can be treated by local excision, i.e., mastoidectomy with tympanoplasty.

A study of some of the ascending and descending vestibular pathways in the pigeon (Columba livia) using anterograde transneuronal autoradiography.

A mixture of tritiated proline and fucose was injected into the endolymph of one of the membranous labyrinths of each of 5 white king pigeons (Columba livia). The membranous labyrinth was resealed and the animals were allowed to survive for 15 days. Brains and upper parts of the spinal cords were sectioned and processed by standard autoradiographic procedures. Clear labeling was noted in structures usually associated with both the ascending auditory pathways and the ascending and descending vestibular pathways. Vestibular structures ipsilateral to the injected labyrinth which contained heavy labeling were Scarpa's ganglion and all 6 vestibular nuclei. No labeling was noted in the contralateral Scarpa's ganglion and sparce, if any, labeling was noted in the contralateral vestibular nuclei. Contralateral structures associated with ascending vestibulo-ocular pathways which contained heavy labeling were the medial longitudinal fasciculus, abducens nucleus, trochlear nucleus, and two parts of the oculomotor nucleus--the dorsolateral part and the ventromedial part. Less heavily labeled ipsilateral vestibulo-ocular-related structures included the medial longitudinal fasciculus, abducens nucleus and the ventrolateral edge of the trochlear nucleus. The dorsomedial part of the oculomotor nucleus was heavily labeled on the side ipsilateral to the injected labyrinth. Slight, if any, labeling was noted in either the ipsilateral or contralateral brachium conjunctivum or regions corresponding to the mammalian ascending tract of Deiters. The medullary core of most folia but primarily the medullary core and granular areas of folia IX and X of the cerebellum were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of three cyanoacrylate glues for ossicular reconstruction.

We evaluated and compared the separate effects of ethyl, isobutyl, and fluoroalkyl cyanoacrylate on the promontory mucosa and surgically disarticulated incudostapedial joint in the adult cat middle ear. The animals were sacrificed at 10-, 30-, and 60-day intervals after glue application. All three cyanoacrylates elicited a chronic inflammatory response when placed directly on the promontory mucosa. The use of ethyl and isobutyl cyanoacrylate resulted in persisting discontinuity of the incudostapedial joint with erosion of the incus. Fluoroalkyl cyanoacrylate maintained incudostapedial continuity without ossicular erosion. Ethyl and isobutyl cyanoacrylate are probably not appropriate for middle ear surgery. The less toxic fluoroalkyl cyanoacrylate may be useful as an ossicular adhesive in selected cases. Our findings are further contrasted with those obtained in similar studies with methyl and butyl cyanoacrylate. The effects of each of the five cyanoacrylates are reviewed in the continuing search for a safe and effective ossicular adhesive.